Optogenetic probes for measuring membrane potential

ABSTRACT

Provided herein are variants of an archaerhodopsin useful for application such as optical measurement of membrane potential. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, cells comprising the polynucleotides, and cells comprising the polypeptides; and methods of using the variants.

RELATED APPLICATIONS

The present application is a continuation of and claims priority under35 U.S.C. §120 to U.S. application Ser. No. 14/742,648, filed Jun. 17,2015, which claims priority under 35 U.S.C. §119(e) to U.S. provisionalpatent application, U.S. Ser. No. 62/013,775, filed Jun. 18, 2014, eachof which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant numbers1-R01-EB012498-01 and IDP2OD007428 awarded by the National Institutes ofHealth (NIH), and under grant number N00014-11-1-0549 awarded by theOffice of Naval Research (ONR). The government has certain rights in theinvention.

BACKGROUND

Membrane-enclosed biological structures can support a voltage differencebetween the inside and the outside of the membrane. This voltage, alsocalled a membrane potential, serves a variety of biological functions,including carrying information (e.g., in neurons), acting as anintermediate in the production of ATP (e.g., in bacteria andmitochondria), powering the flagellar motor (e.g., in bacteria), andcontrolling the transport of nutrients, toxins, and signaling moleculesacross the cell membrane (in bacteria and eukaryotic cells).

In spite of its fundamental biological role, membrane potential is verydifficult to measure. Electrophysiology involves positioning electrodeson both sides of the membrane to record voltage directly.Electrophysiological experiments are slow to set up, can only beperformed on one or a few cells at a time, cannot access deeply buriedtissues (e.g., in vivo), do not work for cells that are too small (e.g.bacteria) or are enclosed in a hard cell wall (e.g. yeast), or aremotile (e.g., sperm), cannot be applied to long-term measurements, andusually damage or kill the cell under study. Accordingly, novel methodsfor measuring membrane potential are needed.

To disentangle the complex interactions underlying neural dynamics, onewould like to visualize membrane voltage across spatial scales, fromsingle dendritic spines to large numbers of interacting neurons, whiledelivering spatially and temporally precise stimuli.^(1,2) Opticalmethods for simultaneous perturbation and measurement of membranepotential could achieve this goal.³ Genetic targeting of the stimulationand recording to genetically specified cells is useful in intact tissuewhere closely spaced cells often perform distinct functions. Genetictargeting in vitro is also useful for characterizing heterogeneouscultures that arise during stem cell differentiation to neurons,⁴ orwhile studying neurons co-cultured with other cell types.

Optical stimulation has been demonstrated with glutamate uncaging,⁵photoactivated ion channel agonists⁶, and microbial rhodopsinactuators.^(7,8) Genetically encoded functional readouts includereporters of intracellular Ca²⁺ and membrane voltage.⁹⁻¹⁴Voltage-sensitive dyes offer good speed, sensitivity, and spectraltuning,^(15,16) but cannot be delivered to a genetically specifiedsubset of cells and often suffer from phototoxicity.

Simultaneous optical stimulation and readout of neural activity havebeen implemented via several combinations of the above techniques.¹⁷⁻²¹However, robust genetically targeted all-optical electrophysiology hasnot been achieved due to limitations on the speed and sensitivity ofgenetically encoded voltage indicators (GEVIs), and spectral overlapbetween existing GEVIs and optogenetic actuators. GFP-based GEVIsexperience severe optical crosstalk with even the most red-shiftedchannelrhodopsins, which retain ˜20% activation with blue lightexcitation. Therefore, there remains a need for sensitive, fast, andspectrally orthogonal tools for genetically targeted simultaneousoptical perturbation and measurement of membrane voltage.

SUMMARY OF THE INVENTION

Provided herein are fluorescent polypeptides which are based on themicrobial rhodopsin family called Archaerhodopsin and are useful asvoltage indicators. The inventive polypeptides provided herein functionin eukaryotic cells such as mammalian cells, e.g., neurons andcardiomyocytes including human stem cell-derived cardiomyocytes. Theinventive polypeptides localize to various cellular locations, e.g., theplasma membrane in eukaryotic cells, and show voltage-dependentfluorescence.

By optically measuring the membrane potential of cells and sub-cellularcompartments, the inventive polypeptides are capable of indicatingelectrical dynamics with sub-millisecond temporal resolution andsub-micron spatial resolution. The inventive polypeptides have improvedproperties over the wild-type Archaerhodopsin such as increasedbrightness, increased sensitivity, higher signal-to-noise ratios,increased linearity with respect to voltage or intensity, and fasterresponse time (increased time resolution), with speed and sensitivitybeing important parameters for evaluating voltage indicators. Theimproved polypeptides provided herein are useful as optically detectablesensors for sensing voltage across membranous structures. It waspreviously demonstrated that the membrane potential in a membranecontaining Archaerhodopsin 3 (Arch 3) can alter the optical propertiesof the protein, thereby making Arch 3 a voltage sensor. The modifiedmicrobial rhodopsin, Arch 3 D95N, has a 40 ms response time and lacksphotoinduced proton pumping. Although the slower response time of thisconstruct hampers detection of membrane potential and changes thereto inneurons, the Arch 3 D95N is fast enough to indicate membrane potentialand action potentials in other types of cells, for example, incardiomyocytes and does not perturb membrane potential in the cellswherein it is used.

Through a combination of directed evolution and targeted mutagenesis,polypeptides based on the human codon-optimized sequence ofAchaerhodopsin genetically encoded voltage indicators GEVIs withimproved performance have been identified.

In certain embodiments, the polypeptide variants are based onArchaerhodopsin such as Archaerhodopsin 3 (Arch 3) and its homologues,including Archaerhodopsin-1, Archaerhodopsin-2, L. Maculans rhodopsin(Mac), Cruxrhodopsin (Crux), and green-absorbing proteorhodopsin (GPR)(see, e.g., Enami et al., J Mol. Biol. (2006) May 5; 358(3):675-85, Epub2006 Mar. 3; Waschuk, S. A. et al., Proc. Natl Acad. Sci. USA (2005)102: 6879-6883; Tateno, M. et al. (1994) Arch. Biochem. Biophys. 315:127-132; Giovannoni et al. (2005) Nature 438(7064): 82-85). Arch 3 hasbeen described in, for example, Chow, B. Y. et al., Nature (2010)463:98-102, which is incorporated herein by reference in its entirety.The inventive polypeptides described herein also include polypeptidesbased on other archaerhopsins with mutations in locations homologous tothose described herein. Other microbial rhodopsins include, but are notlimited to, archaerhodopsin-1 and -2, L. Maculans rhodopsin (Mac),Cruxrhodopsin (Crux), and green-absorbing proteorhodopsin (GPR).

The present invention relates to variants of Archaerhodopsin, comprisingat least one or two amino acid substitutions at positions correspondingto positions P60, T80, D95, D106, or F161 of the archaerhodopsinsequence of SEQ ID NO: 1, wherein the variant has at least 80% but lessthan 100% sequence identity with the archaerhodopsin sequence of SEQ IDNO: 1, and wherein the variant has no proton pumping activity.

The present invention also relates to polynucleotides encoding thepolypeptides; nucleic acid constructs, vectors, cells comprising thepolynucleotides; cells comprising the polypeptides; and methods of usingthe polypeptides and polynucleotides described herein.

Definitions

The inventive polypeptides are generally referred to or described as a“genetically encoded voltage indicator” (GEVI), which is usedinterchangeably with the phrases “voltage-indicating protein” (VIP),“optical sensor”, or “optical voltage indicators”, or similar phrases.As described in more detail herein, the inventive polypeptides employedyield an optical signal indicative of the voltage drop across themembrane in which it is embedded.

The terms “variant” or “mutant” means a polypeptide based on thesequence of archaerhodopsin comprising an alteration, i.e., asubstitution, insertion, and/or deletion, at one or more positions ofthe polypeptide. A substitution means a replacement of an amino acidoccupying a position with a different amino acid; a deletion meansremoval of an amino acid occupying a position; and an insertion meansadding 1-3 amino acids adjacent to an amino acid occupying a position.Variants include those with homologous mutations in another microbialrhodopsin (e.g., another archaerhodopsin) that corresponds to the aminoacid mutations specifically listed herein that is expected to have asimilar effect to a substantially similar mutation in bacteriorhodopsin.One of skill in the art can easily locate a homologous residue in theirdesired microbial rhodopsin by performing an alignment of conservedregions of the desired microbial rhodopsin with a bacteriorhodopsinsequence using a computer program such as ClustalW. Examples ofhomologous mutations include the mutations made in the Examples setforth in this application. The terms variant or mutant also refers to apolynucleotide variant encoding a polypeptide variant described herein.The polynucleotide variant encompasses all forms of mutations includingdeletions, insertions, and point mutations in the coding sequence.

The term “polypeptide” or “polynucleotide” means a polypeptide orpolynucleotide variant that is separate from its native environment,modified by humans, and is present in sufficient quantity to permit itsidentification or use. The polypeptide or polynucleotide is one that isnot part of, or included in its native host. For example, a nucleic acidor polypeptide sequence may be naturally expressed in a cell or organismof a member of Halobacterium sodomense but when the sequence is not partof or included in a Halobacterium sodomense cell or organism, it isconsidered to be isolated. Thus, a polypeptide or polynucleotidesequence of an Archaerhodopsin that is present in a vector, in aheterologous cell, tissue, or organism, etc., is an isolated sequence.The term “heterologous” as used herein, means a cell, tissue or organismthat is not the native cell, tissue, or organism. The polynucleotidesprovided herein may be DNA, RNA, semi-synthetic, synthetic origin, orany combinations thereof.

The term “coding sequence” means a polynucleotide, which directlyspecifies the amino acid sequence of its polypeptide product. Theboundaries of the coding sequence are generally determined by an openreading frame, which usually begins with the ATG start codon oralternative start codons such as GTG and TTG and ends with a stop codonsuch as TAA, TAG, and TGA.

The term “nucleic acid construct” means a nucleic acid molecule, eithersingle- or double-stranded, which is modified to contain segments ofnucleic acids in a manner that would not otherwise exist in nature orwhich is synthetic. The nucleic acid construct may be part of anexpression vector or may be an expression vector when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence of the present invention.

The term “operably linked” means a configuration in which a controlsequence is placed at an appropriate position relative to the codingsequence of a polynucleotide such that the control sequence directs theexpression of the coding sequence.

The term “expression” includes any step involved in the production ofthe polypeptide variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The term “expression vector” means a linear or circular DNA moleculethat comprises a polynucleotide encoding a variant and is operablylinked to additional nucleotides that provide for its expression.

The term “homologous,” as used herein, is an art-understood term thatrefers to nucleic acids or proteins that are highly related at the levelof nucleotide or amino acid sequence. Nucleic acids or proteins that arehomologous to each other are termed homologues. Homologous may refer tothe degree of sequence similarity between two sequences (i.e.,nucleotide sequence or amino acid). The homology percentage figuresreferred to herein reflect the maximal homology possible between twosequences, i.e., the percent homology when the two sequences are soaligned as to have the greatest number of matched (homologous)positions. Homology can be readily calculated by known methods such asthose described in: Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press,1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., andGriffin, H. G., eds., Humana Press, New Jersey, 1994; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; each of which is incorporated herein by reference.Methods commonly employed to determine homology between sequencesinclude, but are not limited to those disclosed in Carillo, H., andLipman, D., SIAM J Applied Math., 48:1073 (1988), incorporated herein byreference. Techniques for determining homology are codified in publiclyavailable computer programs. Exemplary computer software to determinehomology between two sequences include, but are not limited to, GCGprogram package, Devereux, J., et al., Nucleic Acids Research, 12(1),387 (1984)), BLASTP, BLASTN, and PASTA Atschul, S. F. et al., J Molec.Biol., 215, 403 (1990)).

The term “identity” refers to the overall relatedness between nucleicacids (e.g. DNA and/or RNA) or between proteins. Calculation of thepercent identity of two nucleic acid sequences, for example, can beperformed by aligning the two sequences for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondnucleic acid sequences for optimal alignment and non-identical sequencescan be disregarded for comparison purposes). In certain embodiments, thelength of a sequence aligned for comparison purposes is at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or 100% of the length of the referencesequence. The nucleotides at corresponding nucleotide positions are thencompared. When a position in the first sequence is occupied by the samenucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position. The percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences, taking into account the number ofgaps, and the length of each gap, which needs to be introduced foroptimal alignment of the two sequences. The comparison of sequences anddetermination of percent identity between two sequences can beaccomplished using a mathematical algorithm. For example, the percentidentity between two nucleotide sequences can be determined usingmethods such as those described in Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Sequence Analysis in Molecular Biology, von Heinje, G.,Academic Press, 1987; Computer Analysis of Sequence Data, Part I,Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds.,M Stockton Press, New York, 1991; each of which is incorporated hereinby reference. For example, the percent identity between two nucleotidesequences can be determined using the algorithm of Meyers and Miller(CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGNprogram (version 2.0) using a PAM 120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. The percent identity between twonucleotide sequences can, alternatively, be determined using the GAPprogram in the GCG software package using an NWSgapdna.CMP matrix.Methods commonly employed to determine percent identity betweensequences include, but are not limited to those disclosed in Carillo,H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporatedherein by reference. Techniques for determining identity are codified inpublicly available computer programs. Exemplary computer software todetermine homology between two sequences include, but are not limitedto, GCG program package, Devereux, J., et al., Nucleic Acids Research,12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Atschul, S. F. et al., J.Molec. Biol., 215, 403 (1990)).

As used herein, the term “protein” refers to a polymer of at least twoamino acids linked to one another by peptide bonds. The terms, “protein”and “polypeptides” are used interchangeably herein. Proteins may includemoieties other than amino acids (e.g., may be glycoproteins) and/or maybe otherwise processed or modified. Those of ordinary skill in the artwill appreciate that a “protein” can be a complete polypeptide chain asproduced by a cell (with or without a signal sequence), or can be afunctional portion thereof. Those of ordinary skill will furtherappreciate that a protein can sometimes include more than onepolypeptide chain, for example, linked by one or more disulfide bonds orassociated by other means. A polypeptide may refer to an individualpeptide or a collection of polypeptides. Polypeptides may containL-amino acids, D-amino acids, or both and may contain any of a varietyof amino acid modifications or analogs known in the art. Usefulmodifications include, e.g., addition of a chemical entity such as acarbohydrate group, a phosphate group, a farnesyl group, an isofarnesylgroup, a fatty acid group, an amide group, a terminal acetyl group, alinker for conjugation, functionalization, or other modification (e.g.,alpha amidation), etc. In certain embodiments, the modifications of thepeptide lead to a more stable peptide (e.g., greater half-life in vivo).These modifications may include cyclization of the peptide, theincorporation of D-amino acids, etc. None of the modifications shouldsubstantially interfere with the desired biological activity of thepeptide. In certain embodiments, the modifications of the peptide leadto a more biologically active peptide. In certain embodiments,polypeptides may comprise natural amino acids, non-natural amino acids(i.e., compounds that do not occur in nature but that can beincorporated into a peptide chain), synthetic amino acids, amino acidanalogs, and combinations thereof. A polypeptide may be just a fragmentof a naturally occurring protein. A polypeptide may be naturallyoccurring, recombinant, synthetic, or any combination thereof.

“Microbial rhodopsins” are a large class of proteins characterized byseven transmembrane domains and a retinilydene chromophore bound in theprotein core to a lysine via a Schiff base (Beja, O., et al. Nature 411,786-789 (2001)). Over 5,000 microbial rhodopsins are known, and theseproteins are found in all kingdoms of life. Microbial rhodopsins serve avariety of functions for their hosts: some are light-driven proton pumps(bacteriorhodopsin, proteorhodopsins), others are light-driven ionchannels (channelrhodopsins), chloride pumps (halorhodopsins), or servein a purely photosensory capacity (sensory rhodopsins). The retinilydenechromophore imbues microbial rhodopsins with unusual optical properties.The linear and nonlinear responses of the retinal are highly sensitiveto interactions with the protein host: small changes in theelectrostatic environment can lead to large changes in absorptionspectrum. These electro-optical couplings provide the basis for voltagesensitivity in microbial rhodopsins.

In nature, microbial rhodopsins contain a bound molecule of retinalwhich serves as the optically active element. These proteins will alsobind and fold around many other chromophores with similar structure, andpossibly preferable optical properties. Analogues of retinal with lockedrings cannot undergo trans-cis isomerization, and therefore have higherfluorescence quantum yields (Brack et al. Biophys. J. 65, 964-972(1993)). Analogues of retinal with electron-withdrawing substituentshave a Schiff base with a lower pKa than natural retinal and thereforemay be more sensitive to voltage (Sheves et al. Proc. Nat. Acad. Sci.U.S.A. 83, 3262-3266 (1986); Rousso, I., et al. Biochemistry 34,12059-12065 (1995)). Covalent modifications to the retinal molecule maylead to voltage-indicating proteins (VIPs) with significantly improvedoptical properties and sensitivity to voltage.

“Archaerhodopsin 3” (Arch 3 or Ar 3) is a microbial rhodopsin that is alight-driven proton pump found in Halobacterium sodomense (Chow et al.,High-performance genetically targetable optical neural silencing bylight-driven proton pumps. Nature (2010) 463:98-102), capturing solarenergy for its host (Ihara et al., Evolution of the archaeal rhodopsins:evolution rate changes by gene duplication and functionaldifferentiation. J. Mol. Biol. (1999) 285: 163-174). Genbank number:P96787. Arch 3 is an Archaerhodopsin from H. sodomense, and it is knownas a genetically-encoded reagent for high-performance yellow/green-lightneural silencing. Gene sequence at GenBank: GU045593.1 (syntheticconstruct Arch 3 gene).

The term “additional fluorescent molecule” refers to fluorescentproteins other than microbial rhodopsins. Such molecules may include,e.g., green fluorescent proteins and their homologs. Fluorescentproteins that are not microbial rhodopsins are well known and commonlyused, and examples can be found, e.g., in a review, The Family ofGFP-Like Proteins: Structure, Function, Photophysics and BiosensorApplications. Introduction and Perspective, by Rebekka M. Wachter(Photochemistry and Photobiology Volume 82, Issue 2, pages 339-344,March 2006). Also, a review by Nathan C Shaner, Paul A Steinbach, &Roger Y Tsien, entitled A guide to choosing fluorescent proteins (NatureMethods—2, 905-909 (2005)) provides examples of additional usefulfluorescent proteins.

As used herein the phrase “reduced ion pumping activity” means adecrease in the endogenous ion pumping activity of a modified microbialrhodopsin protein of at least 10% compared to the endogenous pumpingactivity of the natural microbial rhodopsin protein from which themodified rhodopsin is derived. The ions most commonly pumped bymicrobial rhodopsins are H⁺ and Cl⁻. In some embodiments, the ionpumping activity of a modified rhodopsin protein is at least 20% lower,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 99% lower than theendogenous ion pumping activity of the corresponding wild type microbialrhodopsin protein. In certain embodiments, the modified microbialrhodopsin has no detectable ion pumping activity.

As used herein, the term “endogenous ion pumping activity” refers to themovement of ions through the wild-type microbial rhodopsin protein thatoccurs in response to light stimuli.

As used herein, the term “wild-type”, “natural”, or “native” microbialrhodopsin protein refers to a rhodopsin protein (e.g., Archaerhodopsin)prepared from a microbial (e.g., bacterial, archaeal, or eukaryotic)source. Such natural microbial rhodopsin proteins, when isolated, retaincharacteristics (e.g., pKa, ion pumping activity, etc.) that aresubstantially similar to the microbial rhodopsin protein in its nativeenvironment (e.g., in a microbial cell). Some non-limiting examples ofmicrobial rhodopsin proteins useful with the methods described hereininclude green-absorbing proteorhodopsin (GPR; GenBank accession numberAF349983), blue-absorbing proteorhodopsin (BPR, GenBank accession numberAF349981), Natromonas pharaonis sensory rhodopsin II (NpSRII; GenBankaccession number Z35086.1), and bacteriorhodopsin (BR; the proteinencoded by GenBank sequence NC_010364.1, nucleotides 1082241-1083029,wherein 1082241 is designated as 1 herein, GenBank accession numberM11720.1, or as described by e.g., Beja et al., (2000). Science 289(5486): 1902-1904), and archaerhodopsin (see e.g., Chow et al., Nature463:98-102 (2010) and the Examples in this application).

As used herein, the term “variant”, “mutant”, or “modified” microbialrhodopsin protein refers to a wild-type microbial rhodopsin proteincomprising at least one mutation. Mutations can be in the nucleic acidsequence (e.g., genomic or mRNA sequence), or alternatively can comprisean amino acid substitution. Such amino acid substitutions can beconserved mutations or non-conserved mutations. As well-known in theart, a “conservative substitution” of an amino acid or a “conservativesubstitution variant” of a polypeptide refers to an amino acidsubstitution which maintains: 1) the structure of the backbone of thepolypeptide (e.g. a beta sheet or alpha-helical structure); 2) thecharge or hydrophobicity of the amino acid; or 3) the bulkiness of theside chain. More specifically, the well-known terminologies “hydrophilicresidues” relate to serine or threonine. “Hydrophobic residues” refer toleucine, isoleucine, phenylalanine, valine or alanine. “Positivelycharged residues” relate to lysine, arginine or histidine. “Negativelycharged residues” refer to aspartic acid or glutamic acid. Residueshaving “bulky side chains” refer to phenylalanine, tryptophan ortyrosine. To avoid doubt as to nomenclature, the term “D97N” or similarterms specifying other specific amino acid substitutions means that theAsp (D) at position 97 of the protein sequence is substituted with Asn(N). A “conservative substitution variant” of D97N would substitute aconservative amino acid variant of Asn (N) that is not D.

The terminology “conservative amino acid substitutions” is well known inthe art, which relates to substitution of a particular amino acid by onehaving a similar characteristic (e.g., similar charge or hydrophobicity,similar bulkiness). Examples include aspartic acid for glutamic acid, orisoleucine for leucine. A list of exemplary conservative amino acidsubstitutions is given in the Table 1 below. A conservative substitutionmutant or variant will 1) have only conservative amino acidsubstitutions relative to the parent sequence, 2) will have at least 90%sequence identity with respect to the parent sequence, generally atleast 95% identity, 96% identity, 97% identity, 98% identity or 99%identity; and 3) will retain voltage sensing activity as that term isdefined herein.

TABLE 1 Conservative Amino Acid Substitutions For Amino Acid CodeReplace With Alanine A D-ala, Gly, Aib, β-Ala, Acp, L-Cys, D-CysArginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met,D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-GlnAspartic D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Acid Cysteine CD-Cys, S—Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn,Glu, D-Glu, Asp, D-Asp Glutamic E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln,D-Gln Acid Glycine G Ala, D-Ala, Pro, D-Pro, Aib, β-Ala, Acp IsoleucineI D-Ile, Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met, D-Met Leucine L D-Leu,Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg,D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-OrnMethionine M D-Met, S—Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenyl-F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, alanine D-Trp, Trans-3,4or 5-phenylproline, AdaA, AdaG, cis-3,4 or 5-phenylproline, Bpa, D-BpaProline P D-Pro, L-I-thioazolidine-4-carboxylic acid, D-or-L-1-oxazolidine-4-carboxylic acid (Kauer, U.S. Pat. No. (4,511,390)Serine D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met (O), D-Met (O),L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met (O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His,D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met, AdaA, AdaGA non-conservative mutation is any other amino acid substitution otherthan the conservative substitutions noted in the above Table 1.

Methods of making conservative amino acid substitutions are also wellknown to one skilled in the art and include but are not limited tosite-specific mutagenesis using oligonucleotide primers and polymerasechain reactions. Optical sensor variants can be expressed and assayedfor voltage sensing activity, pKa, and fluorescence detection by methodsknown in the art and/or described herein to verify that the desiredactivities of the optical sensor are retained or augmented by the aminoacid substitutions. It is contemplated that conservative amino acidsubstitution variants of the optical sensors described herein can haveenhanced activity or superior characteristics for sensing voltagerelative to the parent optical sensor. Certain silent or neutralmissense mutations can also be made in the nucleic acid encoding anoptical sensor by a mutation that does not change the encoded amino acidsequence of the encoded optical sensor. These types of mutations areuseful to optimize codon usage which improve recombinant proteinexpression and production in the desired cell type. Specificsite-directed mutagenesis of a nucleic acid encoding an optical sensorin a vector can be used to create specific amino acid mutations andsubstitutions. Site-directed mutagenesis can be carried out using, e.g.,the QUICKCHANGE® site-directed mutagenesis kit from STRATAGENE®according to manufacture's instructions, or by any method known in theart.

As used herein, the term “membrane potential” refers to a calculateddifference in voltage between the interior and exterior of a cell. Inone embodiment membrane potential, ΔV, is determined by the equationΔV=V_(interior)−V_(exterior). For example, if the outside voltage is 100mV, and the inside voltage is 30 mV, then the difference is −70 mV.Under resting conditions, the membrane potential is predominantlydetermined by the ion having the greatest conductance across themembrane. In many cells, the membrane potential is determined bypotassium, which yields a resting membrane potential of approximately−70 mV. Thus by convention, a cell under resting conditions has anegative membrane potential. In some cells when a membrane potential isreached that is equal to or greater than a threshold potential, anaction potential is triggered and the cell undergoes depolarization(i.e., a large increase in the membrane potential). Often, when a cellundergoes depolarization, the membrane potential reverses and reachespositive values (e.g., 35 mV). During resolution of the membranepotential following depolarization towards the resting membranepotential, a cell can “hyperpolarize.” The term “hyperpolarize” refersto membrane potentials that are more negative than the resting membranepotential, while the term “depolarize” refers to membrane potentialsthat are less negative (or even positive) compared to the restingmembrane potential. Membrane potential changes can arise by movement ofions through ion channels or ion pumps embedded in the membrane.Membrane potential can be measured across any cellular membrane thatcomprises ion channels or ion pumps that can maintain an ionic gradientacross the membrane (e.g., plasma membrane, mitochondrial inner andouter membranes etc.).

As used herein, the term “change in the membrane potential” refers to anincrease (or decrease) in ΔV of at least 1 mV that is either spontaneousor in response to e.g., environmental or chemical stimuli (e.g.,cell-to-cell communication, ion channel modulation, contact with acandidate agent, etc.) compared to the resting membrane potentialmeasured under control conditions (e.g., absence of an agent, impairedcellular communication, etc.). In some embodiments, the membranepotential ΔV is increased by at least 10 mV, at least 15 mV, at least 20mV, at least 25 mV, at least 30 mV, at least 35 mV, at least 40 mV, atleast 45 mV, at least 50 mV, at least 55 mV, at least 60 mV, at least 65mV, at least 70 mV, at least 75 mV, at least 80 mV, at least 85 mV, atleast 90 mV, at least 95 mV, at least 100 mV, at least 105 mV, at least110 mV, at least 115 mV, at least 120 mV, at least 125 mV, at least 130mV, at least 135 mV, at least 140 mV, at least 145 mV, at least 150 mV,at least 155 mV, at least 160 mV, at least 165 V, at least 170 mV, atleast 180 mV, at least 190 mV, at least 200 mV or more compared to themembrane potential of a similar cell under control conditions. In otherembodiments, the membrane potential is decreased by at least 3 mV, atleast 5 mV, at least 10 mV, at least 15 mV, at least 20 mV, at least 25mV, at least 30 mV, at least 35 mV, at least 40 mV, at least 45 mV, atleast 50 mV, at least 55 mV, at least 60 mV, at least 65 mV, at least 70mV, at least 75 mV, at least 80 mV, at least 85 mV, at least 90 mV, atleast 95 mV, at least 100 mV, at least 105 mV, at least 110 mV, at least115 mV, at least 120 mV, at least 125 mV, at least 130 mV, at least 135mV, at least 140 mV, at least 145 mV, at least 150 mV or more comparedto the membrane potential of a similar cell under control conditions.

As used herein, the phrase “localizes to a membrane of the cell” refersto the preferential localization (trafficking) of the modified microbialrhodopsin protein to the membrane of a cell and can be achieved by e.g.,modifying the microbial rhodopsin to comprise a signal sequence thatdirects the rhodopsin protein to a membrane of the cell (e.g., theplasma membrane, the mitochondrial outer membrane, the mitochondrialinner membrane, etc.). In some embodiments, at least 40% of the modifiedmicrobial rhodopsin protein in the cell is localized to the desiredcellular membrane compartment (e.g., plasma membrane, mitochondrialmembrane etc); in other embodiments, at least 50%, at least 60%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 99% of the modified microbial rhodopsin protein islocalized to the desired cellular membrane compartment. Similarly, thephrase “localized to a subcellular compartment” refers to thepreferential localization (trafficking) of the microbial rhodopsinprotein to a particular subcellular compartment (e.g., mitochondria,endoplasmic reticulum, peroxisome etc.). In some embodiments, at least40% of the modified microbial rhodopsin protein in the cell is localizedto the desired subcellular compartment; in other embodiments, at least50%, at least 60%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or at least 99% of the modifiedmicrobial rhodopsin protein is localized to the desired subcellularcompartment. In certain embodiment, about 100% is localized to thedesired cellular membrane or compartment.

As used herein, the term “introducing to a cell” refers to any methodfor introducing either an expression vector encoding an optical sensoror a recombinant optical sensor protein described herein into a hostcell. Some non-limiting examples of introducing an expression vectorinto a cell include, for example, calcium phosphate transfection,electroporation, lipofection, or a method using a gene gun or the like.In one embodiment, a recombinant optical sensor protein is introduced toa cell by membrane fusion using a lipid mediated delivery system, suchas micelles, liposomes, etc.

As used herein, the phrase “a moiety that produces an optical signal”refers to a molecule (e.g., retinal), or moiety of a molecule, capableof producing a detectable signal such as e.g., fluorescence,chemiluminescence, a colorimetric signal etc. In one embodiment, themodified microbial rhodopsin comprises a fusion molecule with a moietythat produces an optical signal.

As used herein, the phrases “change in the level of fluorescence” or “achange in the level of the optical signal” refer to an increase ordecrease in the level of fluorescence from the modified microbialrhodopsin protein or an increase or decrease in the level of the opticalsignal induced by a change in voltage or membrane potential. In someembodiments, the level of fluorescence or level of optical signal in acell is increased by at least at least 2%, at least 5%, at least 10%,20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 99%, at least1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least50-fold, at least 100-fold, at least 500-fold, at least 600-fold, atleast 700-fold, at least 800-fold, at least 900-fold, at least1000-fold, at least 2000-fold, at least 5000-fold, at least 10000-foldor more compared to the same cell or a similar cell under controlconditions. Alternatively, the level of fluorescence or level of opticalsignal in a cell is decreased by at least by at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 99%, or even 100% (i.e., no detectablesignal) compared to the same cell or a similar cell under controlculture conditions.

As used herein, the phrase “modulates ion channel activity” refers to anincrease or decrease in one or more properties of an ion channel thatmanifests as a change in the membrane potential of a cell. Theseproperties include, e.g., open- or closed-state conductivity, thresholdvoltage, kinetics and/or ligand affinity. In some embodiments, the oneor more properties of interest of an ion channel of a cell as measuredby e.g., a change in membrane potential of the cell. In someembodiments, the activity of an ion channel is increased by at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 99%, at least1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or morein the presence of an agent compared to the activity of the ion channelin the absence of the agent. In other embodiments, the parameter ofinterest of an ion channel is decreased by at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or at least 99% in the presence of an agentcompared to the activity of the ion channel in the absence of the agent.In some embodiments, the parameter of an ion channel is absent in thepresence of an agent compared to the activity of the ion channel in theabsence of the agent.

As used herein, the term “targeting sequence” refers to a moiety orsequence that homes to or preferentially associates or binds to aparticular tissue, cell type, receptor, organelle, or other area ofinterest. The addition of a targeting sequence to an optical sensorcomposition will enhance the delivery of the composition to a desiredcell type or subcellular location. The addition to, or expression of, atargeting sequence with the optical sensor in a cell enhances thelocalization of the optical sensor to a desired location within ananimal or subject.

As used herein, the phrase “homologous mutation in another microbialrhodopsin that corresponds to the amino acid mutation inbacteriorhodopsin” refers to mutation of a residue in a desiredmicrobial rhodopsin that is expected to have a similar effect to asubstantially similar mutation in bacteriorhodopsin. One of skill in theart can easily locate a homologous residue in their desired microbialrhodopsin by performing an alignment of conserved regions of the desiredmicrobial rhodopsin with a bacteriorhodopsin sequence using a computerprogram such as ClustalW. Examples of homologous mutations include themutations made in the Examples set forth in this application.

The visible light spectrum ranges from approximately 400 nm toapproximately 750 nm. It is understood in the art that, since light is aspectrum, there will be overlap in wavelengths found between theadjacent colors in the spectrum. Longest visible wavelengths are at thered end of the spectrum. Shortest visible wavelengths are at the blueend of the spectrum. As used herein, “red light” refers to a wavelengthfrom about 600 nm to about 750 nm. As used herein, “orange light” refersto a wavelength from about 580 nm to about 620 nm. As used herein,“yellow light” refers to a wavelength from about 560 nm to about 585 nm.As used herein, “green light” refers to a wavelength from about 500 nmto about 565 nm. As used herein, “blue light” generally refers to awavelength from about 435 nm to about 500 nm. As used herein, “indigolight” generally refers to a wavelength from about 420 nm to about 440nm. As used herein, “violet light” generally refers to a wavelength fromabout 400 nm to about 420 nm. A red-shifted spectrum refers to either anabsorption or emission spectrum towards longer wavelengths (i.e.,towards the red end of the spectrum). A blue-shifted spectrum refers toeither an absorption or emission spectrum towards shorter wavelengths(i.e., towards the blue end of the spectrum).

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the amino acid sequence of the wild-type (WT)Archaerhodopsin 3, also referred to herein as Arch 3 or Ar3.

SEQ ID NO:2 is the amino acid sequence of QuarsAr1 with substitutions atamino acid positions 60, 80, 95, 106, or 161 (positions indicated withunderlining).

SEQ ID NO:3 is the amino acid sequence of QuarsAr2 with substitutions atamino acid positions 60, 80, 95, 106, or 161 (positions indicated withunderlining).

SEQ ID NO:4 is an exemplary polynucleotide sequence that encodeswild-type (WT) Archaerhodopsin 3.

SEQ ID NO:5 is an exemplary polynucleotide sequence that encodesQuarsAr1.

SEQ ID NO:6 is an exemplary polynucleotide sequence that encodesQuarsAr2.

SEQ ID NO:7 is the trafficking sequence (TS).

SEQ ID NO:8 is the endoplasmic reticulum (ER) export motif from Kir2.1(FCYENE).

SEQ ID NO:9 is an exemplary polynucleotide sequence that encodestrafficking sequence (TS).

SEQ ID NO:10 is an exemplary polynucleotide sequence that endoplasmicreticulum (ER) export motif

TABLE 2 SEQ ID Name: Sequence NO: Arch 3MDPIALQAGYDLLGDGRPETLWLGIGTLLMLIGTFYFLVRGWG  1 (WT)VTDKDAREYYAVTILVPGIASAAYLSMFFGIGLTEVTVGGEMLDIYYARYADWLFTTPLLLLDLALLAKVDRVTIGTLVGVDALMIVTGLIGALSHTAIARYSWWLFSTICMIVVLYFLATSLRSAAKERGPEVASTFNTLTALVLVLWTAYPILWIIGTEGAGVVGLGIETLLFMVLDVTAKVGFGFILLRSRAILGDTEAPEPSAGADVSAAD QuasAr1MDPIALQAGYDLLGDGRPETLWLGIGTLLMLIGTFYFLVRGWG  2VTDKDAREYYAVTILVSGIASAAYLSMFFGIGLTEVSVGGEMLDIYYARYAHWLFTTPLLLLHLALLAKVDRVTIGTLVGVDALMIVTGLIGALSHTAIARYSWWLFSTICMIVVLYVLATSLRSAAKERGPEVASTFNTLTALVLVLWTAYPILWIIGTEGAGVVGLGIETLLFMVLDVTAKVGFGFILLRSRAILGDTEAPEPSAGADVSAAD QuasAr2MDPIALQAGYDLLGDGRPETLWLGIGTLLMLIGTFYFLVRGWG  3VTDKDAREYYAVTILVSGIASAAYLSMFFGIGLTEVSVGGEMLDIYYARYAQWLFTTPLLLLHLALLAKVDRVTIGTLVGVDALMIVTGLIGALSHTAIARYSWWLFSTICMIVVLYVLATSLRSAAKERGPEVASTFNTLTALVLVLWTAYPILWIIGTEGAGVVGLGIETLLFMVLDVTAKVGFGFILLRSRAILGDTEAPEPSAGADVSAAD Arch 3ATGGACCCCATCGCTCTGCAGGCTGGTTACGACCTGCT  4 (WT)GGGTGACGGCAGACCTGAAACTCTGTGGCTGGGCATCGGCACTCTGCTGATGCTGATTGGAACCTTCTACTTTCTGGTCCGCGGATGGGGAGTCACCGATAAGGATGCCCGGGAATATTACGCTGTGACTATCCTGGTGCCCGGAATCGCATCCGCCGCATATCTGTCTATGTTCTTTGGTATCGGGCTTACTGAGGTGACCGTCGGGGGCGAAATGTTGGATATCTATTATGCCAGGTACGCCGACTGGCTGTTTACCACCCCACTTCTGCTGCTGGATCTGGCCCTTCTCGCTAAGGTGGATCGGGTGACCATCGGCACCCTGGTGGGTGTGGACGCCCTGATGATCGTCACTGGCCTCATCGGAGCCTTGAGCCACACGGCCATAGCCAGATACAGTTGGTGGTTGTTCTCTACAATTTGCATGATAGTGGTGCTCTATTTTCTGGCTACATCCCTGCGATCTGCTGCAAAGGAGCGGGGCCCCGAGGTGGCATCTACCTTTAACACCCTGACAGCTCTGGTCTTGGTGCTGTGGACCGCTTACCCTATCCTGTGGATCATAGGCACTGAGGGCGCTGGCGTGGTGGGCCTGGGCATCGAAACTCTGCTGTTTATGGTGTTGGACGTGACTGCCAAGGTCGGCTTTGGCTTTATCCTGTTGAGATCCCGGGCTATTCTGGGCGACACCGAGGCACCAGAACCCAGTGCCGGTGCCGA TGTCAGTGCCGCCGACTAA QuasAr1ATGGACCCCATCGCTCTGCAGGCTGGITACGACCTGCTGGG  5TGACGGCAGACCTGAAACTCTGTGGCTGGGCATCGGCACTCTGCTGATGCTGATTGGAACCTTCTACTTTCTGGTCCGCGGATGGGGAGTCACCGATAAGGATGCCCGGGAATATTACGCTGTGACTATCCTGGTGTCNGGAATCGCATCCGCCGCATATCTGTCTATGTTCTTTGGTATCGGGCTTACTGAGGTGTCNGTCGGGGGCGAAATGTTGGATATCTATTATGCCAGGTACGCCCAYTGGCTGTTTACCACCCCACTTCTGCTGCTGCAYCTGGCCCTTCTCGCTAAGGTGGATCGGGTGACCATCGGCACCCTGGTGGGTGTGGACGCCCTGATGATCGTCACTGGCCTCATCGGAGCCTTGAGCCACACGGCCATAGCCAGATACAGTTGGTGGTTGTTCTCTACAATTTGCATGATAGTGGTGCTCTATGTNCTGGCTACATCCCTGCGATCTGCTGCAAAGGAGCGGGGCCCCGAGGTGGCATCTACCTTTAACACCCTGACAGCTCTGGTCTTGGTGCTGTGGACCGCTTACCCTATCCTGTGGATCATAGGCACTGAGGGCGCTGGCGTGGTGGGCCTGGGCATCGAAACTCTGCTGTTTATGGTGTTGGACGTGACTGCCAAGGTCGGCTTTGGCTTTATCCTGTTGAGATCCCGGGCTATTCTGGGCGACACCGAGGCACCAGAACCCAGTGCCGGTGCCGATGTCAGTGCCGCCGACTAA QuasAr1ATGGACCCCATCGCTCTGCAGGCTGGTTACGACCTGCTGGG 28TGACGGCAGACCTGAAACTCTGTGGCTGGGCATCGGCACTCTGCTGATGCTGATTGGAACCTTCTACTTTCTGGTCCGCGGATGGGGAGTCACCGATAAGGATGCCCGGGAATATTACGCTGTGACTATCCTGGTGTCNGGAATCGCATCCGCCGCATATCTGTCTATGTTCTTTGGTATCGGGCTTACTGAGGTGAGYGTCGGGGGCGAAATGTTGGATATCTATTATGCCAGGTACGCCCAYTGGCTGTTTACCACCCCACTTCTGCTGCTGCAYCTGGCCCTTCTCGCTAAGGTGGATCGGGTGACCATCGGCACCCTGGTGGGTGTGGACGCCCTGATGATCGTCACTGGCCTCATCGGAGCCTTGAGCCACACGGCCATAGCCAGATACAGTTGGTGGTTGTTCTCTACAATTTGCATGATAGTGGTGCTCTATGTNCTGGCTACATCCCTGCGATCTGCTGCAAAGGAGCGGGGCCCCGAGGTGGCATCTACCTTTAACACCCTGACAGCTCTGGTCTTGGTGCTGTGGACCGCTTACCCTATCcrGTGGATCATAGGCACTGAGGGCGCTGGCGTGGTGGGCCTGGGCATCGAAACTCTGCTGTTTATGGTGTTGGACGTGACTGCCAAGGTCGGCTTTGGCTTTATCCTGTTGAGATCCCGGGCTATTCTGGGCGACACCGAGGCACCAGAACCCAGTGCCGGTGCCGATGTCAGTGCCGCCGACTAA QuasAr1ATGGACCCCATCGCTCTGCAGGCTGGTTACGACCTGCTGGG 29TGACGGCAGACCTGAAACTCTGTGGCTGGGCATCGGCACTCTGCTGATGCTGATTGGAACCTTCTACTTTCTGGTCCGCGGATGGGGAGTCACCGATAAGGATGCCCGGGAATATTACGCTGTGACTATCCTGGTGAGYGGAATCGCATCCGCCGCATATCTGTCTATGTTCTTTGGTATCGGGCTTACTGAGGTGTCNGTCGGGGGCGAAATGTTGGATATCTATTATGCCAGGTACGCCCAYTGGCTGTTTACCACCCCACTTCTGCTGCTGCAYCTGGCCCTTCTCGCTAAGGTGGATCGGGTGACCATCGGCACCCTGGTGGGTGTGGACGCCCTGATGATCGTCACTGGCCTCATCGGAGCCTTGAGCCACACGGCCATAGCCAGATACAGTTGGTGGTTGTTCTCTACAATTTGCATGATAGTGGTGCTCTATGTNCTGGCTACATCCCTGCGATCTGCTGCAAAGGAGCGGGGCCCCGAGGTGGCATCTACCTTTAACACCCTGACAGCTCTGGTCTTGGTGCTGTGGACCGCTTACCCTATCCTGTGGATCATAGGCACTGAGGGCGCTGGCGTGGTGGGCCTGGGCATCGAAACTCTGCTGTTTATGGTGTTGGACGTGACTGCCAAGGTCGGCTTTGGCTTTATCCTGTTGAGATCCCGGGCTATTCTGGGCGACACCGAGGCACCAGAACCCAGTGCCGGTGCCGATGTCAGTGCCGCCGACTAA QuasAr1ATGGACCCCATCGCTCTGCAGGCTGGTTACGACCTGCTGGG 30TGACGGCAGACCTGAAACTCTGTGGCTGGGCATCGGCACTCTGCTGATGCTGATTGGAACCTTCTACTTTCTGGTCCGCGGATGGGGAGTCACCGATAAGGATGCCCGGGAATATTACGCTGTGACTATCCTGGTGAGYGGAATCGCATCCGCCGCATATCTGTCTATGTTCTTTGGTATCGGGCTTACTGAGGTGAGYGTCGGGGGCGAAATGTTGGATATCTATTATGCCAGGTACGCCCAYTGGCTGTTTACCACCCCACTTCTGCTGCTGCAYCTGGCCCTTCTCGCTAAGGTGGATCGGGTGACCATCGGCACCCTGGTGGGTGTGGACGCCCTGATGATCGTCACTGGCCTCATCGGAGCCTTGAGCCACACGGCCATAGCCAGATACAGTTGGTGGTTGTTCTCTACAATTTGCATGATAGTGGTGCTCTATGTNCTGGCTACATCCCTGCGATCTGCTGCAAAGGAGCGGGGCCCCGAGGTGGCATCTACCTTTAACACCCTGACAGCTCTGGTCTTGGTGCTGTGGACCGCTTACCCTATCCTGTGGATCATAGGCACTGAGGGCGCTGGCGTGGTGGGCCTGGGCATCGAAACTCTGCTGTTTATGGTGTTGGACGTGACTGCCAAGGTCGGCTTTGGCTTTATCCTGTTGAGATCCCGGGCTATTCTGGGCGACACCGAGGCACCAGAACCCAGTGCCGGTGCCGATGTCAGTGCCGCCGACTAA QuasAr2ATGGACCCCATCGCTCTGCAGGCTGGTTACGACCTGCTGGG  6TGACGGCAGACCTGAAACTCTGTGGCTGGGCATCGGCACTCTGCTGATGCTGATTGGAACCTTCTACTTTCTGGTCCGCGGATGGGGAGTCACCGATAAGGATGCCCGGGAATATTACGCTGTGACTATCCTGGTGTCNGGAATCGCATCCGCCGCATATCTGTCTATGTTCTTTGGTATCGGGCTTACTGAGGTGTCNGTCGGGGGCGAAATGTTGGATATCTATTATGCCAGGTACGCCCARTGGCTGTTTACCACCCCACTTCTGCTGCTGCAYCTGGCCCTTCTCGCTAAGGTGGATCGGGTGACCATCGGCACCCTGGTGGGTGTGGACGCCCTGATGATCGTCACTGGCCTCATCGGAGCCTTGAGCCACACGGCCATAGCCAGATACAGTTGGTGGTTGTTCTCTACAATTTGCATGATAGTGGTGCTCTATGTNCTGGCTACATCCCTGCGATCTGCTGCAAAGGAGCGGGGCCCCGAGGTGGCATCTACCTTTAACACCCTGACAGCTCTGGTCTTGGTGCTGTGGACCGCTTACCCTATCCTGTGGATCATAGGCACTGAGGGCGCTGGCGTGGTGGGCCTGGGCATCGAAACTCTGCTGTTTATGGTGTTGGACGTGACTGCCAAGGTCGGCTTTGGCTTTATCCTGTTGAGATCCCGGGCTATTCTGGGCGACACCGAGGCACCAGAACCCAGTGCCGGTGCCGATGTCAGTGCCGCCGACTAA QuasAr2ATGGACCCCATCGCTCTGCAGGCTGGTTACGACCTGCTGGG 31TGACGGCAGACCTGAAACTCTGTGGCTGGGCATCGGCACTCTGCTGATGCTGATTGGAACCTTCTACTTTCTGGTCCGCGGATGGGGAGTCACCGATAAGGATGCCCGGGAATATTACGCTGTGACTATCCTGGTGTCNGGAATCGCATCCGCCGCATATCTGTCTATGTTCTTTGGTATCGGGCTTACTGAGGTGAGYGTCGGGGGCGAAATGTTGGATATCTATTATGCCAGGTACGCCCARTGGCTGTTTACCACCCCACTTCTGCTGCTGCAYCTGGCCCTTCTCGCTAAGGTGGATCGGGTGACCATCGGCACCCTGGTGGGTGTGGACGCCCTGATGATCGTCACTGGCCTCATCGGAGCCTTGAGCCACACGGCCATAGCCAGATACAGTTGGTGGTTGTTCTCTACAATTTGCATGATAGTGGTGCTCTATGTNCTGGCTACATCCCTGCGATCTGCTGCAAAGGAGCGGGGCCCCGAGGTGGCATCTACCTTTAACACCCTGACAGCTCTGGTCTTGGTGCTGTGGACCGCTTACCCTATCCTGTGGATCATAGGCACTGAGGGCGCTGGCGTGGTGGGCCTGGGCATCGAAACTCTGCTGTTTATGGTGTTGGACGTGACTGCCAAGGTCGGCTTTGGCTTTATCCTGTTGAGATCCCGGGCTATTCTGGGCGACACCGAGGCACCAGAACCCAGTGCCGGTGCCGATGTCAGTGCCGCCGACTAA QuasAr2ATGGACCCCATCGCTCTGCAGGCTGGTTACGACCTGCTGGG 32TGACGGCAGACCTGAAACTCTGTGGCTGGGCATCGGCACTCTGCTGATGCTGATTGGAACCTTCTACTTTCTGGTCCGCGGATGGGGAGTCACCGATAAGGATGCCCGGGAATATTACGCTGTGACTATCCTGGTGAGYGGAATCGCATCCGCCGCATATCTGTCTATGTTCTTTGGTATCGGGCTTACTGAGGTGTCNGTCGGGGGCGAAATGTTGGATATCTATTATGCCAGGTACGCCCARTGGCTGTTTACCACCCCACTTCTGCTGCTGCAYCTGGCCCTTCTCGCTAAGGTGGATCGGGTGACCATCGGCACCCTGGTGGGTGTGGACGCCCTGATGATCGTCACTGGCCTCATCGGAGCCTTGAGCCACACGGCCATAGCCAGATACAGTTGGTGGTTGTTCTCTACAATTTGCATGATAGTGGTGCTCTATGTNCTGGCTACATCCCTGCGATCTGCTGCAAAGGAGCGGGGCCCCGAGGTGGCATCTACCTTTAACACCCTGACAGCTCTGGTCTTGGTGCTGTGGACCGCTTACCCTATCCTGTGGATCATAGGCACTGAGGGCGCTGGCGTGGTGGGCCTGGGCATCGAAACTCTGCTGTTTATGGTGTTGGACGTGACTGCCAAGGTCGGCTTTGGCTTTATCCTGTTGAGATCCCGGGCTATTCTGGGCGACACCGAGGCACCAGAACCCAGTGCCGGTGCCGATGTCAGTGCCGCCGACTAA QuasAr2ATGGACCCCATCGCTCTGCAGGCTGGTTACGACCTGCTGGG 33TGACGGCAGACCTGAAACTCTGTGGCTGGGCATCGGCACTCTGCTGATGCTGATTGGAACCTTCTACTTTCTGGTCCGCGGATGGGGAGTCACCGATAAGGATGCCCGGGAATATTACGCTGTGACTATCCTGGTGAGYGGAATCGCATCCGCCGCATATCTGTCTATGTTCTTTGGTATCGGGCTTACTGAGGTGAGYGTCGGGGGCGAAATGTTGGATATCTATTATGCCAGGTACGCCCARTGGCTGTTTACCACCCCACTTCTGCTGCTGCAYCTGGCCCTTCTCGCTAAGGTGGATCGGGTGACCATCGGCACCCTGGTGGGTGTGGACGCCCTGATGATCGTCACTGGCCTCATCGGAGCCTTGAGCCACACGGCCATAGCCAGATACAGTTGGTGGTTGTTCTCTACAATTTGCATGATAGTGGTGCTCTATGTNCTGGCTACATCCCTGCGATCTGCTGCAAAGGAGCGGGGCCCCGAGGTGGCATCTACCTTTAACACCCTGACAGCTCTGGTCTTGGTGCTGTGGACCGCTTACCCTATCCTGTGGATCATAGGCACTGAGGGCGCTGGCGTGGTGGGCCTGGGCATCGAAACTCTGCTGTTTATGGTGTTGGACGTGACTGCCAAGGTCGGCTTTGGCTTTATCCTGTTGAGATCCCGGGCTATTCTGGGCGACACCGAGGCACCAGAACCCAGTGCCGGTGCCGATGTCAGTGCCGCCGACTAA Trafficking SRITSEGEYIPLNIDINVGG  7sequence ER export FCYENE  8 motif from Kir2.1 Traffickingagtagaatcacaagcgaagacgagtacatccccctggatcaaataaacataaatgtaggtgga  9sequence ER export ttttgttatgagaatga 10 motif from Kir2.1 *can be TCN orAGY where N is any nucleic acid, Y is t/u or c; R is g or a

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings are not intended to be drawn to scale. In theDrawings, for purposes of clarity, not every component may be labeled inevery drawing.

FIGS. 1A-1H show non-pumping Arch-derived voltage indicators withimproved speed, sensitivity, and brightness. FIG. 1A shows thehierarchical screen to select improved Arch mutants. Five rounds ofrandom mutagenesis and screening for brightness were performed in E.coli. The brightest mutants were subjected to targeted mutagenesis andscreening for speed and voltage sensitivity in HeLa cells via inducedtransient voltage (FIG. 2). FIG. 1B shows the fluorescence of Archmutants fused to eGFP and expressed in HEK cells, as a function ofillumination intensity. The plot shows Arch fluorescence (640 nm exc.,660-760 nm em.) normalized by illumination intensity (640 nm) and byeGFP fluorescence (488 nm exc., 525-575 nm em.) to control forcell-to-cell variations in expression. A linear fluorophore (i.e.,brightness proportional to illumination intensity) would appear as ahorizontal line. Error bars represent s.e.m. (n=5-7 cells for eachmutant). FIG. 1D shows fluorescence vs. membrane voltage for Arch,QuasAr1, and QuasAr2 expressed in HEK cells. FIG. 1D shows fluorescenceresponses to a step in membrane voltage from −70 to +30 mV. FIG. 1Eshows simultaneous optical and electrical recording of APs in a rathippocampal neuron expressing QuasAr1. Frame rate 1 kHz. FIG. 1F showsthe overlay of mean optically and electrically recorded AP waveforms.Frame rate 2 kHz. FIGS. 1G and H show the same as FIGS. 1E and 1F inneurons expressing QuasAr2. Data in FIGS. 1B-H acquired on a 128×128pixel EMCCD camera (see Examples).

FIGS. 2A-2D show induced transmembrane voltage (ITV) in Arch-expressingHeLa cells. FIG. 2A shows the experimental setup, showing two platinumelectrodes placed on either side of a transfected cell. V(t) representsthe pulse generator and high-voltage amplifier. FIG. 2B shows framesfrom a movie of a HeLa cell expressing QuasAr1. The cell was stimulatedwith an electrical pulse (20 ms, 50 V/cm). The images show thefluorescence response (ΔF/F). The arrow labeled ‘E’ indicates thedirection of the electric field. FIG. 2C shows fluorescence of the cellpoles during the ITV experiment shown in FIG. 2B. Gray marks above thefluorescence traces indicate timing and duration of the ITV pulses. FIG.2D shows the zoomed in view of one fluorescence intensity peak from FIG.2C.

FIGS. 3A-3D show the structural and spectroscopic properties of QuasArs.FIG. 3A shows the locations of mutations in QuasAr1, modeled on thecrystal structure of Arch-2 (PDB: 2EI4)⁷⁰. Arch-2 has 90% amino acididentity with Arch-3. The retinal chromophore is colored blue andmutations are colored green. FIG. 3B (top) shows images of E. colipellets expressing Arch, QuasAr1, and QuasAr2. FIG. 3B (bottom) showsimages of solubilized protein. FIG. 3C shows the absorption spectra ofArch, QuasAr1 and QuasAr2, measured on solubilized protein. FIG. 3Dshows the excitation and emission spectra measured on QuasAr1 andQuasAr2. Arch was too dim to measure in the fluorimeter. Emissionspectra were recorded with λ_(exc)=600 nm. Excitation spectra weremeasured with λ_(em)=750 nm.

FIGS. 4A-4B show the photophysics of QuasArs in mammalian cells. FIG. 4Ashows wild-type Arch, expressed in cultured rat hippocampal neurons,generated substantial photocurrent of 220±30 pA (n=6 cells) under red(Is, 640 nm, 300 W/cm2) and 140±25 pA under blue (1 s, 488 nm, 500mW/cm²) illumination (n=5 cells). Steady state photocurrents werecalculated by averaging the current over the last 0.25 seconds of lightexposure and subtracting the holding current (cells held at −65 mV) inthe dark. These currents hyperpolarized cells by 25±4 mV and 19±3 mV,respectively. Neither QuasAr1 (n=9 cells) nor QuasAr2 (n=7 cells)generated detectable photocurrents under either illumination condition,nor under red illumination at up to 900 W/cm². FIG. 4 B shows aComparison of fluorescence between QuasAr mutants and Arch doublemutants, expressed as eGFP fusions in HEK cells. The double mutants hadmutations at the locations of the proton acceptor (Asp95) and protondonor (Asp106) to the Schiff base. QuasAr1 includes mutations D95H,D106H, and QuasAr2 includes mutations D95Q, D106H. The three additionalbackbone mutations in the QuasArs (P60S, T80S, F161V) increasedbrightness relative to the double mutants. Fluorescence of each Archmutant was measured with excitation at 640 nm and emission from 660-760nm. To control for variation in expression level, fluorescence wasnormalized by eGFP fluorescence (λ_(exc)=488 nm, λ_(em)=510-550 nm).Error bars represent s.e.m. for measurements on n=5-10 cells.

FIGS. 5A-5B show the quantification of optical crosstalk of blueillumination into QuasAr fluorescence. Illumination sufficient to inducehigh-frequency trains of action potentials (488 nm, 140 mW/cm2)perturbed fluorescence of QuasArs by <1%. FIG. 5A shows the effect ofblue illumination on QuasAr fluorescence. HEK293 cells expressingQuasAr1 or QuasAr2 were exposed to continuous excitation at 640 nm (300W/cm²) and pulses of illumination at 488 nm (50 ms, 5 Hz). The intensityof the blue pulses increased from 0.06 to 1.8 W/cm². FIG. 5B showsquantification of crosstalk. Illumination with blue light at typicalintensity used to excite a blue-light activator (CheRiff) (0.2 W/cm²)increased QuasAr1 fluorescence by 1.1% and QuasAr2 fluorescence by 0.6%.Illumination at with blue light at 1 W/cm² increased QuasAr1fluorescence by 3.4% and QuasAr2 fluorescence by 2.4%. Error barsrepresent s.e.m. for n=5 cells for each QuasAr.

FIG. 6 shows the mechanism of voltage-dependent fluorescence ineFRET-based voltage indicators. Images to the left show cartoons of theprotein structure in a cell plasma membrane. Images to the right showthe voltage-dependent absorption spectrum of the microbial rhodopsin(dashed line) and emission spectrum of the attached fluorescent protein.FIG. 6A is at depolarizing (positive) membrane voltage; the microbialrhodopsin absorbs strongly and quenches the fluorescence of thefluorescent protein. FIG. 6B is at hyperpolarizing (negative) membranevoltage, the microbial rhodopsin absorbs weakly, so the fluorescentprotein emits strongly.

FIGS. 7A-7C show the voltage-indicating properties of five eFRET GEVIsspanning the visible spectrum. FIG. 7A shows images of HEK293 cellsexpressing the eFRET fusion of the indicated fluorescent protein toQuasAr2. Scale bar 10 μm. FIG. 7B shows fluorescence as a function ofvoltage. FIG. 7C shows the fluorescence response to a step in membranevoltage from −70 mV to +30 mV.

FIGS. 8A-8C show single-trial detection of neuronal action potentialswith three eFRET GEVIs. FIG. 8A shows images of cultured rat hippocampalneurons expressing eFRET GEVIs. FIG. 8B shows simultaneous patch clampelectrophysiology and fluorescence recordings of neuronal actionpotentials. FIG. 8C shows close-ups showing the action potentialwaveform as reported by patch clamp electrophysiology and as reported bythe eFRET GEVI.

FIGS. 9A-9E shows a comparison of voltage-indicating properties ofQuasArs and ArcLight A242 in culture. FIG. 9A shows fluorescence as afunction of membrane voltage in HEK293T cells. ArcLight showed voltagesensitivity of −32±3% ΔF/F per 100 mV (n=7 cells), comparable inmagnitude to QuasAr1 and 2.8-fold smaller than QuasAr2. FIG. 9B showsthe response of ArcLight to steps in membrane voltage. ArcLight showedbi-exponential kinetics in response to rising or falling voltage steps(Table 6). Mean half-response times were 42±8 ms and 76±5 ms on risingand falling edges at 23° C. (n=6 cells) and 11±1 and 17±2 ms on risingand falling edges at 34° C. (n=7 cells). FIG. 9C shows step responses ofArcLight and QuasArs overlaid on the same time axis at 23° C. (top) and34° C. (bottom). FIG. 9D shows Continuous illumination of a neuronexpressing ArcLight (488 nm, 10 W/cm²) led to photobleaching with a timeconstant of 70 s. Inset: Low-magnification image of the neuron. Scalebar 20 μm. Cyan box shows field of view used for high-speed (1 kHz framerate) movies of fluorescence dynamics. Fluorescence was calculated bypreferentially weighting the pixels whose intensity co-varies with thewhole-field average. FIG. 9E shows single-trial fluorescence response ofArcLight and QuasAr2 to a single AP, recorded at 34° C. and a 1 kHzframe rate. ArcLight reported action potentials with an amplitude ofΔF/F=−2.7±0.5% (n=5 cells) and a single-trial signal-to-noise ratio(SNR) of 8.8±1.6 (488 nm, 10 W/cm²). ArcLight distorted the AP waveformsto have a width of 14.5±3.0 ms at 70% maximal fluorescence deviation,compared to the true width of 1.3±0.1 ms simultaneously recorded with apatch pipette. QuasAr2 reported APs at 34° C. and 23° C. with comparablesingle-trial SNR (SNR at 34° C.: 41±3, 300 W/cm², n=8 cells).

FIG. 10 shows the extraction of fluorescence traces from QuasAr movies.Fluorescence can either be calculated by manually defining a region ofinterest (ROI; top row), or by preferentially weighting the pixels whoseintensity co-varies with the whole-field average (bottom row)¹⁰. Thenoise in the fluorescence trace when scaled to match the electricalrecording is denoted σ_(v). With the improved trafficking of the QuasArmutants compared to Arch, the automated technique gave only slightlyhigher SNR than manual definition of the ROI. The technique makes no useof the electrode readout. Cell shown is the source of the data in FIG.1G. All comparisons of SNR in culture were made on measurements takenwith the same 60× objective, collected on the same EMCCD, and extractedusing this automated technique. For recordings on cultured neurons,values of ΔF/F were calculated after subtracting backgroundautofluorescence from a cell-free region of the field of view. Thisbackground subtraction was not performed on recordings in tissue.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofimproved mutants of a microbial rhodopsin proteins (e.g.,Archaerhodopsin) or modified microbial rhodopsin proteins that havereduced ion pumping activity, compared to the wild type microbialrhodopsin protein from which they are derived. The polypeptides providedherein can be used as an optically detectable sensor to sense voltageacross membranous structures, such as cells and sub-cellular organelles.That is, the polypeptides provided herein can be used as voltage sensorsto measure changes in membrane potential of cells and sub-cellularorganelles, including prokaryotic and eukaryotic cells. The opticalsensors described herein are not constrained by the need for electrodesand permit electrophysiological studies to be performed in, e.g.,subcellular compartments (e.g., mitochondria) or in small cells (e.g.,bacteria). The optical sensors described herein can be used in methodsfor drug screening, in research settings, and in in vivo imagingsystems. The voltage indicators are generally referred to as geneticallyencoded voltage indicators (GEVIs).

Table 3 shows exemplary approximate characteristics of fluorescentvoltage indicating proteins and contains representative members of thefamilies of fluorescent indicators.

TABLE 3 Representative members of families of fluorescent indicators.Approx Approx- ΔF/F imate per response Molecule 100 mV time CommentsVSFP 2.3, Knopfel, T. 9.5% 78 ms Ratio- et al. J. Neurosci. 30, metric14998-15004 (2010) (ΔR/R) VSFP 2.4 Knopfel, T. 8.9% 72 ms Ratio- et al.J. Neurosci. 30, metric 14998-15004 (2010) (ΔR/R) VSFP 3.1, Lundby, A.,et  3% 1-20 ms Protein al., PLoS One 3, 2514 (2008) Mermaid, Perron, A.et 9.2% 76 Ratio- al. Front Mol Neurosci. metric 2, 1-8 (2009) (ΔR/R)SPARC, Ataka, K. & 0.5% 0.8 ms Protein Pieribone, V. A. Biophys. J. 82,509- 516 (2002) Flash, Siegel, M. S. & 5.1% 2.8-85 ms Protein Isacoff,E. Y. Neuron 19, 735-741 (1997) PROPS, described in US 150%  5 msProtein Patent Application No. 2013/0224756 Arch 3 WT, described in  66%<0.5 ms Protein US Patent Application No. 2013/0224756 Arch D95N,described in 100%  41 ms Protein US Patent Application No. 2013/0224756

Generally, GEVIs should have at least one or more of the followinggeneral attributes including:

-   -   High speed: The reporter generally should not distort the        waveform of action potentials in the cells. The action        potentials depends on the cells being measured. For example,        action potentials that rise and fall in less than 0.5 ms, 0.1 ms        or 1 ms.    -   High sensitivity: The reporter generally exhibits a large change        in fluorescence over the physiological voltage range (−70 mV to        +30 mV). In certain cases, the change in fluorescent is linear.    -   High brightness and photostability: For high-speed imaging, many        photons are generally recorded in a short interval. The reporter        should generally maintain a stable level of baseline        fluorescence throughout an experiment.    -   Efficient trafficking to and uniform distribution throughout the        plasma membrane: reproters caught in internal structures        contributes to background fluorescence and noise, but not to        voltage sensitivity.    -   Absence of perturbation to endogenous neuronal dynamics: the        reporter should generally preserve membrane electrical        parameters, and generally should not affect expression or        trafficking of other membrane proteins, patterns of gene        expression, or cellular metabolism or physiology.    -   Far red excitation and emission spectra: Compared to blue light        (typically used to excite GFP), red light offers:    -   far lower tissue autofluorescence: Brain autofluorescence,        dominated by FAD-containing proteins, has excitation and        emission spectral that are nearly indistinguishable from GFP;    -   better tissue penetration: Photons propagate through brain        tissue with a mean free path of d˜λ^(−2.3), where λ is the        wavelength. Excitation light at 640 nm propagates nearly twice        as far as excitation light at 488 nm.    -   lower phototoxicity. On account of fewer endogenous chromophores        at the red end of the spectrum, red excitation tends to preserve        cell health better than blue excitation.        Improved voltage indicators are useful in disease modeling,        using various cells, such as but not limited to primary and        human iPS and ES-derived cells; and in studies of intact tissue        in, for example, mice, zebrafish, C. elegans, and Drosophila        fruit flies. In certain embodiments, the cells are neurons or        cardiomyocytes. Studies using the protein reporter,        Archaerhodopsin 3 (Arch), indicated that Arch is a fast and        sensitive voltage indicator¹ but had properties that could be        improved upon.² Other GEVIs are based on fusion of transmembrane        voltage-sensing domains to fluorescent proteins such as GFP. In        some of these, voltage modulates the brightness of a single        fluorescent fusion,^(3,4) while in others, voltage modulates the        efficiency of fluorescence resonance energy transfer (FRET)        between a pair of fluorescent fusions.^(5,6) Fluorescent        protein-based voltage sensors tend to have high brightness, but        limited speed and sensitivity, and photobleaching can be a        concern. Thus, there is strong demand for improved GEVIs.

Provided herein are polypeptides useful as genetically encoded voltageindicators (GEVIs). As used herein, the inventive polypeptides are alsoreferred to as GEVIs. In certain embodiments, the polypeptides arevariants of archaerhodopsin. In certain embodiments, the polypeptidesare variants of archaerhodopsin 3 (Arch) (SEQ ID NO: 1). In certainembodiments, the polypeptides are variants of an archaerhodopsin-basedvoltage indicator. The polypeptides provided herein are brighter thanArch with a brightness that is a linear function of illuminationintensity.

The polypeptides provided herein were identified using directedevolution (using, e.g., error-prone PCR or PCR DNA shuffling) of Archvariants. About five rounds of directed evolution were used to preparethe Arch mutant library, followed by random mutagenesis. Site-directedmutagenesis is then used to further identify mutants with improvedvoltage sensitivity and speed. Using the foregoing methods, mutations ofamino acids distant from the retinal chromophore were identified thatresulted in polypeptides with improved brightness. In certainembodiments, the polypeptides comprise a C-terminal endoplasmicreticulum (ER) export motif and a trafficking sequence (TS). The TScomprises the amino acid sequence SRITSEGEYIPLDQIDINVGG (SEQ ID NO: 7),wherein the amino acid K is optionally found at the N-terminal end ofthe sequence. The ER comprises the amino acid sequence FCYENE (SEQ IDNO: 8), wherein the amino acid V is optionally found at the C-terminalend of the sequence. An exemplary nucleic acid coding sequence for theTS sequence is:agtagaatcacaagcgaaggcgagtacatccccctggatcaaatagacataaatgtaggtgga (SEQ IDNO: 9), wherein the sequence optionally comprises the nucleotides aag atthe 5′-end that encodes for the optional K residue. An exemplary nucleicacid coding sequence for the ER sequence is: ttttgttatgagaatgaa (SEQ IDNO: 10), wherein the sequence optionally comprises nucleotides gtg atthe 3′-end that encodes for the optional V residue.

Polypeptides and Polynucleotides

The polypeptides provided herein are derived from archaerhodopsinmodified to reduce or inhibit the light-induced ion pumping activity.Thus, the polypeptides and polynucleotides encoding the polypeptidesprovided herein are non-naturally occurring. Such modifications permitthe modified Archaerhodopsin to sense voltage without altering themembrane potential of the cell with its native ion pumping activity andthus altering the voltage of the system. It is contemplated herein thatother archaerhodopsin protein or variants thereof can be engineered asdescribed herein to serve as voltage-indicating proteins.

In certain embodiments, the polypeptides described herein are based onarchaerhodopsin-3 (Arch 3). Mutation of D95 in Arch3 reduces or inhibitsion pumping activity. Other mutations impart other advantageousproperties to the archaerhodopsin-based GEVIs, including increasedfluorescence brightness, improved photostability, tuning of thesensitivity and dynamic range of the voltage response, increasedresponse speed, and tuning of the absorption and emission spectra. Aminoacids at positions 95 and 106 are associated with the protontranslocation during photocycle and at least one amino acid at position60, 80, or 161 of Arch 3 were are associated with improved propertiessuch as brightness. The amino acid at position 60 is in close proximityto the Schiff base and is likely involved in directly influencing thephotophysical properties of the GEVIs. Thus, in certain embodiments, theamino acid at position 60 is mutated to provide increased brightness.The inventive polypeptides herein have a red-shifted absorption andfluorescence spectrum, with minimal overlap with other reporters such aschannelrhodopsin actuators and GFP-based reporters.

The starting sequences from which these inventive polypeptides can beengineered are based on archaerhodopsin protein or variant thereof. Incertain embodiments, the voltage sensor is selected from anarchaerhodopsin protein or variant thereof that provides avoltage-induced shift in its absorption or fluorescence.

Provided herein is a polypeptide comprising an amino acid sequence ofwild-type archaerhodopsin 3 (SEQ ID NO: 1), wherein at least one of theamino acids at positions 60, 80, 95, 106, or 161 has been mutated. Incertain embodiments, the polypeptide comprises an amino acid sequence ofSEQ ID NO: 1, wherein at least two of the amino acids at positions 60,80, 95, 106, or 161 have been mutated. In certain embodiments, thepolypeptide comprises an amino acid sequence of SEQ ID NO: 1, wherein atleast three of the amino acids at positions 60, 80, 95, 106, or 161 havebeen mutated. In certain embodiments, the polypeptide comprises an aminoacid sequence of SEQ ID NO: 1, wherein at least four of the amino acidsat positions 60, 80, 95, 106, or 161 have been mutated. In certainembodiments, the polypeptide comprises an amino acid sequence of SEQ IDNO: 1, wherein at the amino acids at positions 60, 80, 95, 106, or 161have been mutated.

Provided herein is an polypeptide comprising an amino acid sequence ofSEQ ID NO: 1, wherein the amino acid at position 60 is P or S, the aminoacid at position 80 is T or S, the amino acid at position 95 is Asn,His, Gin, Cys, or Tyr, the amino acid at position 106 is Asn, Cys, Gin,Met, Ser, Thr, Asp, Glu, His or Lys, and the amino acid at position 161is Phe or Val. Also provided herein are polynucleotides that encode thepolypeptide.

In certain embodiments, the amino acids involved in proton translocationare mutated. Such mutations around the proton translocation networkaround the Schiff base could affect the voltage sensitivity, responsekinetics, or other photophysical aspects of the GEVIs. In certainembodiments, the brightness of the GEVIs is improved relative to thewild-type protein. For example, amino acids at position 95 or position106 of SEQ ID NO: 1 can be mutated. The amino acids corresponding toamino acids at position 95 or position 106 in another Archaerhodopsin orother microbial rhodopsins can also be similarly mutated.

In certain embodiments, the amino acid at position 60 is P. In certainembodiments, the amino acid at position 60 is S. In certain embodiments,the amino acid at position 80 is T. In certain embodiments, the aminoacid at position 80 is S. In certain embodiments, the amino acid atposition 95 is Asn, His, Gin, Cys, or Tyr. In certain embodiments, theamino acid at position 95 is Asn, Gin, or Cys. In certain embodiments, aAsn, Gin, or Cys at position 95 improves the voltage sensitivity. Incertain embodiments, the amino acid at position 95 is Asn. In certainembodiments, the amino acid at position 95 is His. In certainembodiments, the amino acid at position 95 is Gin. In certainembodiments, the amino acid at position 95 is Cys. In certainembodiments, the amino acid at position 95 is Tyr. In certainembodiments, the amino acid at position 106 is Asn. In certainembodiments, the amino acid at position 106 is Cys. In certainembodiments, the amino acid at position 106 is Gin. In certainembodiments, the amino acid at position 106 is Met. In certainembodiments, the amino acid at position 106 is Ser. In certainembodiments, the amino acid at position 106 is Thr. In certainembodiments, the amino acid at position 106 is Asp. In certainembodiments, the amino acid at position 106 is Glu. In certainembodiments, the amino acid at position 106 is His. In certainembodiments, His at position 106 improves the voltage sensitivity andfast kinetics. In certain embodiments, the amino acid at position 106 isLys. In certain embodiments, the amino acid at position 95 is His andthe amino acid at position 106 is His. In certain embodiments, the aminoacid at position 95 is Gin and the amino acid at position 106 is His. Incertain embodiments, the amino acid at position 95 is either His or Gin.

Mutations that eliminate ion pumping in the inventive polypeptidesgenerally comprise mutations to the Schiff base counterion, specificallya carboxylic amino acid (Asp) conserved on the third transmembrane helix(helix C) of archaerhodopsin. The amino acid sequence is RYX(DE) where Xis a non-conserved amino acid. Mutations of the carboxylic amino aciddirectly affect the proton conduction pathway, eliminating the protonpumping property of the archaerhodopsin. The conserved Asp is located atposition 95 of the Arch 3 amino acid sequence or variants thereof.Polypeptide variants that are at least about 80% homologous or at leastabout 80% identical to the polypeptides herein are contemplated to bewithin the scope of the invention. Thus, for polypeptide variantswherein the conserved Asp is not located at position 95 due to, forexample, additions or deletions in the amino acid sequence, one ofordinary skill in the art would understand that the Asp in thepolypeptide variant to be mutated for purposes of eliminating protonpumping is the Asp in the polypeptide variant that corresponds to theconserved Asp95 of the wild-type Arch 3.

To eliminate proton pumping, the conserved Asp is typically mutated toAsn or Gin, although other mutations are possible such as to a His. Incertain embodiments, the inventive polypeptide comprises thesubstitution of the conserved Asp to Asn, Gin, or His in the Arch 3amino acid sequence. In certain embodiments, the conserved Asp islocated at position 95 of the Arch 3 amino acid sequence. In certainembodiments, the inventive polypeptide comprises the substitution of theconserved Asp to Asn in the Arch 3 amino acid sequence. In certainembodiments, the conserved Asp is located at position 95 of the Arch 3amino acid sequence. In certain embodiments, the inventive polypeptidecomprises the substitution of the conserved Asp to Gin in the Arch 3amino acid sequence. In certain embodiments, the conserved Asp islocated at position 95 of the Arch 3 amino acid sequence in the Arch 3amino acid sequence. In certain embodiments, the inventive polypeptidecomprises the substitution of the conserved Asp to His in the Arch 3amino acid sequence. In certain embodiments, the conserved Asp islocated at position 95 of the Arch 3 amino acid sequence. In certainembodiments, the inventive polypeptide comprises substitution of theconserved Asp to Cys in the Arch 3 amino acid sequence. In certainembodiments, the conserved Asp is located at position 95 of the Arch 3amino acid sequence.

In certain embodiments, the polypeptide comprises an amino acid sequenceof SEQ ID NO: 1, wherein the amino acid sequence comprises a mutation atposition 95, resulting in the polypeptide having reduced ion pumpingactivity compared to a wild type member of the archaerhodopsin family ofproteins from which it is derived. In certain embodiments, the aminoacid at position 95 is mutated from a Asp to His, Gln, Cys, or Asn. Incertain embodiments, the amino acid at position 95 is mutated from a Aspto Gln, Cys or Asn. In certain embodiments, the amino acid at position95 is mutated from a Asp to Gin. In certain embodiments, the amino acidat position 95 is mutated from a Asp to Cys. In certain embodiments, theamino acid at position 95 is mutated from a Asp to Asn, wherein thepolypeptide has an additional mutation as described herein. In certainembodiments where the amino acid at position 95 is mutated from a Asp toAsn, the polypeptide has at least one mutation at an amino acid residueselected from positions 60, 80, 106, and 161.

Provided herein are also polypeptides based on the amino acid sequenceof wild-type archaerhodopsin 3. In certain embodiments, the polypeptidecomprises an amino acid sequence of wild-type archaerhodopsin 3 (SEQ IDNO: 1), wherein the amino acid sequence for the polypeptide comprises atleast one mutation selected from P60S, T80S, D95H, D106H, and F161V. Incertain embodiments, the amino acid sequence for the polypeptidecomprises at least two mutations selected from P60S, T80S, D95H, D106H,and F161V. In certain embodiments, the amino acid sequence for thepolypeptide comprises at least three mutations selected from P60S, T80S,D95H, D106H, and F161V. In certain embodiments, the amino acid sequencefor the polypeptide comprises at least four mutations selected fromP60S, T80S, D95H, D106H, and F161V. In certain embodiments, the aminoacid sequence for the polypeptide comprises at least five mutationsselected from P60S, T80S, D95H, D106H, and F161V. In certainembodiments, the polypeptide comprises an amino acid sequence of SEQ IDNO: 2.

In certain embodiments, the polypeptide comprises an amino acid sequenceof wild-type archaerhodopsin 3 (SEQ ID NO: 1), wherein the amino acidsequence for the polypeptides comprises at least one mutation selectedfrom P60S, T80S, D95Q, D106H, and F161V. In certain embodiments, thepolypeptide comprises an amino acid sequence of SEQ ID NO: 1 comprisingat least two mutations selected from P60S, T80S, D95Q, D106H, and F161V.In certain embodiments, the polypeptide comprises an amino acid sequenceof SEQ ID NO: 1 comprising at least three mutations selected from P60S,T80S, D95Q, D106H, and F161V. In certain embodiments, the polypeptidecomprises an amino acid sequence of SEQ ID NO: 1 comprising at leastfour mutations selected from P60S, T80S, D95Q, D106H, and F161V. Incertain embodiments, the polypeptide comprises an amino acid sequence ofSEQ ID NO: 1 comprising the P60S, T80S, D95Q, D106H, and F161Vmutations. In certain embodiments, the polypeptide comprises an aminoacid sequence of SEQ ID NO: 3.

In certain embodiments, the polypeptide comprises an amino acid sequenceof SEQ ID NO: 1, wherein the amino acid sequence comprises a mutation atposition 95, and at least one mutation selected from P60S, T80S, D106H,and F161V. In certain embodiments, the polypeptide comprises an aminoacid sequence of SEQ ID NO: 1, wherein the amino acid sequence comprisesa mutation at position 95, and at least two mutations selected fromP60S, T80S, D106H, and F161V. In certain embodiments, the polypeptidecomprises an amino acid sequence of SEQ ID NO: 1, wherein the amino acidsequence comprises a mutation at position 95 and least three mutationsselected from P60S, T80S, D106H, and F161V. In certain embodiments, thepolypeptide comprises an amino acid sequence of SEQ ID NO: 1, whereinthe amino acid sequence comprises a mutation at position 95 and theP60S, T80S, D106H, and F161V mutations.

In certain embodiments, the polypeptide comprises an amino acid sequenceof SEQ ID NO: 1, wherein the amino acid sequence comprises a mutation atposition 106 to a polar or charged amino acid. In certain embodiments,the polypeptide comprises an amino acid sequence of SEQ ID NO: 1,wherein the amino acid sequence comprises a mutation at position 106 toa polar amino acid selected from Asn, Cys, Gin, Met, Ser, and Thr. Incertain embodiments, the polypeptide comprises an amino acid sequence ofSEQ ID NO: 1, wherein the amino acid sequence comprises a mutation atposition 106 to a charged amino acid selected from Asp and Glu. Incertain embodiments, the polypeptide comprises an amino acid sequence ofSEQ ID NO: 1, wherein the amino acid sequence comprises a mutation atposition 106 to a charged amino acid selected from Arg, His, and Lys. Incertain embodiments, the polypeptide comprises an amino acid sequence ofSEQ ID NO: 1, wherein the amino acid sequence comprises a mutation atposition 106 to His.

In certain embodiments, the polypeptide comprises an amino acid sequenceof SEQ ID NO: 1, wherein the amino acid sequence comprises a mutation atposition 106, and at least one mutation selected from P60S, T80S,D95(N/H/Q/C), and F161V. In certain embodiments, the polypeptidecomprises an amino acid sequence of SEQ ID NO: 1, wherein the amino acidsequence comprises a mutation at position 106 and a mutation at position60. In certain embodiments, position 106 is mutated to His. In certainembodiments, the polypeptide comprises an amino acid sequence of SEQ IDNO: 1, wherein the amino acid sequence comprises a mutation at position106, and at least two mutations selected from P60S, T80S, D95(N/H/Q/C),and F161V. In certain embodiments, position 106 is mutated to His. Incertain embodiments, the polypeptide comprises an amino acid sequence ofSEQ ID NO: 1, wherein the amino acid sequence comprises a mutation atposition 106, and at least three mutation selected from P60S, T80S,D95(N/H/Q/C), and F161V. In certain embodiments, position 106 is mutatedto His. In certain embodiments, the polypeptide comprises an amino acidsequence of SEQ ID NO: 1, wherein the amino acid sequence comprises amutation at position 106 and the P60S, T80S, D106H, and F161V mutations.In certain embodiments, position 106 is mutated to His.

In certain embodiments, the polypeptide comprises an amino acid sequenceof SEQ ID NO: 1, wherein the amino acid sequence comprises a mutation atposition 95, a mutation at position 106, and at least one mutationselected from P60S, T80S, and F161V. In certain embodiments, thepolypeptide comprises an amino acid sequence of SEQ ID NO: 1, whereinthe amino acid sequence comprises a mutation at position 95, a mutationat position 106, and least two mutations selected from P60S, T80S, andF161V. In certain embodiments, the polypeptide comprises an amino acidsequence of SEQ ID NO: 1, wherein the amino acid sequence comprises amutation at position 95, a mutation at position 106, and the P60S, T80S,or F161V mutations. In certain embodiments, the amino acid at position95 is mutated to Asn, His, Gin, Cys, or Tyr. In certain embodiments, theamino acid at position 95 is mutated to Asn, Gin, or Cys. In certainembodiments, the amino acid at position 95 is mutated to His. In certainembodiments, the amino acid at position 95 is mutated to Gln. In certainembodiments, the amino acid at position 106 is mutated to His or Tyr. Incertain embodiments, the amino acid at position 106 is mutated to His.In certain embodiments, the amino acid at position 106 is mutated toTyr.

In certain embodiments, the polypeptide comprising an amino acidsequence of SEQ ID NO: 1, wherein the amino acid sequence comprises amutation at position 106 and least one mutation selected from P60S,T80S, and F161V. In certain embodiments, the polypeptide comprising anamino acid sequence of SEQ ID NO: 1, wherein the amino acid sequencecomprises a mutation at position 106 and least two mutation selectedfrom P60S, T80S, and F161V. In certain embodiments, the polypeptidecomprising an amino acid sequence of SEQ ID NO: 1, wherein the aminoacid sequence comprises a mutation at position 106 and the P60S, T80S,and F161V mutations. In certain embodiments, the amino acid at position106 is mutated to His or Tyr. In certain embodiments, the amino acid atposition 106 is mutated to His. In certain embodiments, the amino acidat position 106 is mutated to Tyr. In certain embodiments, thepolypeptide comprising an amino acid sequence of SEQ ID NO: 1, whereinthe amino acid sequence comprises a His at position 106 and least onemutation selected from P60S, T80S, and F161V.

In certain embodiments, the polypeptide comprises an amino acid sequenceof SEQ ID NO: 1, wherein the amino acid sequence comprises a mutation atposition 95, a mutation at position 106, and a mutation at position 60.In certain embodiments, the polypeptide comprises an amino acid sequenceof SEQ ID NO: 1, wherein the amino acid sequence comprises a mutation atposition 95, a mutation at position 106, a mutation at position 60, andleast one mutation from T80S or F161V. In certain embodiments, thepolypeptide comprises an amino acid sequence of SEQ ID NO: 1, whereinthe amino acid sequence comprises a mutation at position 95, a mutationat position 106, a mutation at position 60, and the T80S or F161Vmutations. In certain embodiments, the amino acid at position 95 is His,the amino acid at position 106 is His, and the amino acid at position 60is Ser.

Provided herein is a polypeptide comprising an amino acid sequence ofSEQ ID NO: 2. SEQ ID NO: 2 differs from the sequence of the wild-typeArch 3 with respect to the following mutations: P60S, T80S, D95H, D106H,and F161V. Also contemplated is a polypeptide variant of SEQ ID NO: 2comprising the P60S, T80S, D95H, D106H, and F161V mutations butcomprises an alteration, i.e., a substitution, insertion, and/ordeletion, at one or more other positions of the polypeptide.Polypeptides that are homologous to SEQ ID NO: 2 are also contemplated.

In certain embodiments, the polypeptide comprises a sequence that is atleast about 80% homologous to the amino acid sequence of SEQ ID NO: 2.In certain embodiments, the polypeptide comprises a sequence that is atleast about 85% homologous to the amino acid sequence of SEQ ID NO: 2.In certain embodiments, the polypeptide comprises a sequence that is atleast about 90% homologous to the amino acid sequence of SEQ ID NO: 2.In certain embodiments, the polypeptide comprises a sequence that is atleast about 95% homologous to the amino acid sequence of SEQ ID NO: 2.In certain embodiments, the polypeptide comprises a sequence that is atleast about 96% homologous to the amino acid sequence of SEQ ID NO: 2.In certain embodiments, the polypeptide comprises a sequence that is atleast about 97% homologous to the amino acid sequence of SEQ ID NO: 2.In certain embodiments, the polypeptide comprises a sequence that is atleast about 98% homologous to the amino acid sequence of SEQ ID NO: 2.In certain embodiments, the polypeptide comprises a sequence that is atleast about 99% homologous to the amino acid sequence of SEQ ID NO: 2.In certain embodiments, the polypeptide comprises a sequence that is atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% homologous to the amino acid sequence of SEQ ID NO: 2.

In certain embodiments, the polypeptide comprises a sequence that is atleast about 80% identical to the amino acid sequence of SEQ ID NO: 2. Incertain embodiments, the polypeptide comprises a sequence that is atleast about 85% identical to the amino acid sequence of SEQ ID NO: 2. Incertain embodiments, the polypeptide comprises a sequence that is atleast about 90% identical to the amino acid sequence of SEQ ID NO: 2. Incertain embodiments, the polypeptide comprises a sequence that is atleast about 95% identical to the amino acid sequence of SEQ ID NO: 2. Incertain embodiments, the polypeptide comprises a sequence that is atleast about 96% identical to the amino acid sequence of SEQ ID NO: 2. Incertain embodiments, the polypeptide comprises a sequence that is atleast about 97% identical to the amino acid sequence of SEQ ID NO: 2. Incertain embodiments, the polypeptide comprises a sequence that is atleast about 98% identical to the amino acid sequence of SEQ ID NO: 2. Incertain embodiments, the polypeptide comprises a sequence that is atleast about 99% identical to the amino acid sequence of SEQ ID NO: 2. Incertain embodiments, the polypeptide comprises a sequence that is atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% identical to the amino acid sequence of SEQ ID NO: 2.

Provided herein is a polypeptide comprising an amino acid sequence ofSEQ ID NO: 3. SEQ ID NO: 3 differs from the sequence of the wild-typeArch 3 with respect to the following mutations: P60S, T80S, D95Q, D106H,and F161V. Also contemplated is a polypeptide variant of SEQ ID NO: 3comprising the P60S, T80S, D95Q, D106H, and F161V mutations butcomprises an alteration, i.e., a substitution, insertion, and/ordeletion, at one or more other positions of the polypeptide.Polypeptides that are homologous to SEQ ID NO: 3 are also contemplated.

In certain embodiments, the polypeptide comprises a sequence that is atleast about 80% homologous to the amino acid sequence of SEQ ID NO: 3.In certain embodiments, the polypeptide comprises a sequence that is atleast about 85% homologous to the amino acid sequence of SEQ ID NO: 3.In certain embodiments, the polypeptide comprises a sequence that is atleast about 90% homologous to the amino acid sequence of SEQ ID NO: 3.In certain embodiments, the polypeptide comprises a sequence that is atleast about 95% homologous to the amino acid sequence of SEQ ID NO: 3.In certain embodiments, the polypeptide comprises a sequence that is atleast about 96% homologous to the amino acid sequence of SEQ ID NO: 3.In certain embodiments, the polypeptide comprises a sequence that is atleast about 97% homologous to the amino acid sequence of SEQ ID NO: 3.In certain embodiments, the polypeptide comprises a sequence that is atleast about 98% homologous to the amino acid sequence of SEQ ID NO: 3.In certain embodiments, the polypeptide comprises a sequence that is atleast about 99% homologous to the amino acid sequence of SEQ ID NO: 3.In certain embodiments, the polypeptide comprises a sequence that is atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% homologous to the amino acid sequence of SEQ ID NO: 3.

In certain embodiments, the polypeptides comprise a sequence that is atleast about 80% identical to the amino acid sequence of SEQ ID NO: 3. Incertain embodiments, the polypeptide comprises a sequence that is atleast about 85% identical to the amino acid sequence of SEQ ID NO: 3. Incertain embodiments, the polypeptide comprises a sequence that is atleast about 90% identical to the amino acid sequence of SEQ ID NO: 3. Incertain embodiments, the polypeptide comprises a sequence that is atleast about 95% identical to the amino acid sequence of SEQ ID NO: 3. Incertain embodiments, the polypeptide comprises a sequence that is atleast about 96% identical to the amino acid sequence of SEQ ID NO: 3. Incertain embodiments, the polypeptide comprises a sequence that is atleast about 97% identical to the amino acid sequence of SEQ ID NO: 3. Incertain embodiments, the polypeptide comprises a sequence that is atleast about 98% identical to the amino acid sequence of SEQ ID NO: 3. Incertain embodiments, the polypeptide comprises a sequence that is atleast about 99% identical to the amino acid sequence of SEQ ID NO: 3. Incertain embodiments, the polypeptide comprises a sequence that is atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 96%, at least about 97%, at least about 98%, or atleast about 99% identical to the amino acid sequence of SEQ ID NO: 3.

In certain embodiments, the polypeptides are encoded by a nucleic acidsequence that is at least about 80% identical to the nucleic acidsequence of SEQ ID NO: 5. In certain embodiments, the polypeptides areencoded by a nucleic acid sequence that is at least about 85% identicalto the nucleic acid sequence of SEQ ID NO: 5. In certain embodiments,the polypeptides are encoded by a nucleic acid sequence that is at leastabout 90% identical to the nucleic acid sequence of SEQ ID NO: 5. Incertain embodiments, the polypeptides are encoded by a nucleic acidsequence that is at least about 95% identical to the nucleic acidsequence of SEQ ID NO: 5. In certain embodiments, the polypeptides areencoded by a nucleic acid sequence that is at least about 96% identicalto the nucleic acid sequence of SEQ ID NO: 5. In certain embodiments,the polypeptides are encoded by a nucleic acid sequence that is at leastabout 97% identical to the nucleic acid sequence of SEQ ID NO: 5. Incertain embodiments, the polypeptides are encoded by a nucleic acidsequence that is at least about 98% identical to the nucleic acidsequence of SEQ ID NO: 5. In certain embodiments, the polypeptides areencoded by a nucleic acid sequence that is at least about 99% identicalto the nucleic acid sequence of SEQ ID NO: 5. In certain embodiments,the polypeptides are encoded by a nucleic acid sequence that is at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% identical to the nucleic acid sequence of SEQ ID NO: 5.

In certain embodiments, the polypeptides are encoded by a nucleic acidsequence that is at least about 80% identical to the nucleic acidsequence of SEQ ID NO: 6. In certain embodiments, the polypeptides areencoded by a nucleic acid sequence that is at least about 85% identicalto the nucleic acid sequence of SEQ ID NO: 6. In certain embodiments,the polypeptides are encoded by a nucleic acid sequence that is at leastabout 90% identical to the nucleic acid sequence of SEQ ID NO: 6. Incertain embodiments, the polypeptides are encoded by a nucleic acidsequence that is at least about 95% identical to the nucleic acidsequence of SEQ ID NO: 6. In certain embodiments, the polypeptides areencoded by a nucleic acid sequence that is at least about 96% identicalto the nucleic acid sequence of SEQ ID NO: 6. In certain embodiments,the polypeptides are encoded by a nucleic acid sequence that is at leastabout 97% identical to the nucleic acid sequence of SEQ ID NO: 6. Incertain embodiments, the polypeptides are encoded by a nucleic acidsequence that is at least about 98% identical to the nucleic acidsequence of SEQ ID NO: 6. In certain embodiments, the polypeptides areencoded by a nucleic acid sequence that is at least about 99% identicalto the nucleic acid sequence of SEQ ID NO: 6. In certain embodiments,the polypeptides are encoded by a nucleic acid sequence that is at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at leastabout 99% identical to the nucleic acid sequence of SEQ ID NO: 6.

Also contemplated are nucleic acid sequences that are homologous to thenucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. Two nucleotidesequences are considered to be homologous if the polypeptides theyencode are at least about 50% identical, at least about 60% identical,at least about 70% identical, at least about 80% identical, at leastabout 90% identical, or at least about 95% identical for at least onestretch of at least 20 amino acids. Generally, homologous nucleotidesequences are also characterized by the ability to encode a stretch ofat least 4-5 uniquely specified amino acids. Both the identity and theapproximate spacing of these amino acids relative to one another areconsidered for nucleotide sequences to be considered homologous. Forexample, nucleotide sequences less than 60 nucleotides in length,homology is determined by the ability to encode a stretch of at leastabout 4-5 uniquely specified amino acids.

In certain embodiments, the polypeptides provided herein have deletions,substitutions, and/or additions of 1 to 25 amino acids. In certainembodiments, the polypeptides provided herein have deletions,substitutions, and/or additions of 1 to 5 amino acids. In certainembodiments, the polypeptides provided herein have deletions,substitutions, and/or additions of 5 to 10 amino acids. In certainembodiments, the polypeptides provided herein have deletions,substitutions, and/or additions of 10 to 15 amino acids. In certainembodiments, the polypeptides provided herein have deletions,substitutions, and/or additions of 15 to 20 amino acids. In certainembodiments, the polypeptides provided herein have deletions,substitutions, and/or additions of 20 to 25 amino acids.

Provided herein are polynucleotides encoding any inventive polypeptideprovided herein. Also provided herein are polynucleotides encoding apolypeptide comprising an amino acid sequence of SEQ ID NO: 2 or variantthereof. Further provided herein are polynucleotides encoding apolypeptide comprising an amino acid sequence of SEQ ID NO: 3 or variantthereof.

With respect to polynucleotide sequences herein, degeneracy of thegenetic code provides the possibility to substitute at least one base ofthe base sequence of a gene with a different base without causing theamino acid sequence of the polypeptide produced from the gene to bechanged. Hence, the polynucleotides of the present invention may alsohave any base sequence that has been changed from a sequence recitedherein, e.g., in Table 2, by substitution in accordance with degeneracyof genetic code. References describing codon usage include Carels et al.(1998) J. Mol. Evol., 46:45 and Fennoy et al. (1993) Nuci. Acids Res.21(23):5294.

Provided herein are polynucleotide comprising an nucleic acid sequenceof SEQ ID NO: 4, or complement thereof, wherein the nucleic acidsequence encodes a polypeptide with at least one mutation selected frompositions 60, 80, 95, 106, and 161. In certain embodiments, thepolynucleotide comprising an nucleic acid sequence of SEQ ID NO: 4,wherein the nucleic acid sequence encodes a polypeptide with at leasttwo mutations selected from positions 60, 80, 95, 106, and 161. Incertain embodiments, the polynucleotide comprising an nucleic acidsequence of SEQ ID NO: 4, wherein the nucleic acid sequence encodes apolypeptide with at least three mutations selected from positions 60,80, 95, 106, and 161. In certain embodiments, the polynucleotidecomprising an nucleic acid sequence of SEQ ID NO: 4, wherein the nucleicacid sequence encodes a polypeptide with at least four mutationsselected from positions 60, 80, 95, 106, and 161. In certainembodiments, the polynucleotide comprising an nucleic acid sequence ofSEQ ID NO: 4, wherein the nucleic acid sequence encodes a polypeptidewith mutations at positions 60, 80, 95, 106, and 161.

In certain embodiments, the polynucleotide comprises an nucleic acidsequence of SEQ ID NO: 4, or the complement thereof, wherein the nucleicacid sequence encodes a polypeptide with at least one mutations selectedfrom P60S, T80S, D95(H/Q), D106H, and F161V. In certain embodiments, thepolynucleotide comprises a nucleic acid sequence of SEQ ID NO: 4,wherein the nucleic acid sequence encodes a polypeptide with at leasttwo mutations selected from P60S, T80S, D95(H/Q), D106H, and F161V. Incertain embodiments, the polynucleotide comprises an nucleic acidsequence of SEQ ID NO: 4, wherein the nucleic acid sequence encodes apolypeptide with at least three mutations selected from P60S, T80S,D95(H/Q), D106H, and F161V. In certain embodiments, the polynucleotidecomprises an nucleic acid sequence of SEQ ID NO: 4, wherein the nucleicacid sequence encodes a polypeptide with at least four mutationsselected from P60S, T80S, D95(H/Q), D106H, and F161V. In certainembodiments, the polynucleotide comprises a nucleic acid sequence of SEQID NO: 4, wherein the nucleic acid sequence encodes a polypeptide withthe mutations P60S, T80S, D95H, D106H, and F161V. In certainembodiments, the polynucleotide comprises a nucleic acid sequence of SEQID NO: 4, wherein the nucleic acid sequence encodes a polypeptide withthe mutations P60S, T80S, D95Q, D106H, and F161V.

Provided herein are polynucleotides comprising a nucleic acid sequenceof SEQ ID NO: 5, or the complement thereof. In certain embodiments,polynucleotides comprises a nucleic acid sequence that is at least about80% identical to the nucleic acid sequence of SEQ ID NO: 5. In certainembodiments, polynucleotides comprises a nucleic acid sequence that isat least about 85% identical to the nucleic acid sequence of SEQ ID NO:5. In certain embodiments, polynucleotides comprises a nucleic acidsequence that is at least about 90% identical to the nucleic acidsequence of SEQ ID NO: 5. In certain embodiments, polynucleotidescomprises a nucleic acid sequence that is at least about 95% identicalto the nucleic acid sequence of SEQ ID NO: 5. In certain embodiments,polynucleotides comprises a nucleic acid sequence that is at least about97% identical to the nucleic acid sequence of SEQ ID NO: 5. In certainembodiments, polynucleotides comprises a nucleic acid sequence that isat least about 98% identical to the nucleic acid sequence of SEQ ID NO:5. In certain embodiments, polynucleotides comprises a nucleic acidsequence that is at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 97%, at least about 98%, or atleast about 99% identical to the nucleic acid sequence of SEQ ID NO: 5.

Provided herein are polynucleotides comprising a nucleic acid sequenceof SEQ ID NO: 6, or the complement thereof. In certain embodiments,polynucleotides comprises a nucleic acid sequence that is at least about80% identical to the nucleic acid sequence of SEQ ID NO: 6. In certainembodiments, polynucleotides comprises a nucleic acid sequence that isat least about 85% identical to the nucleic acid sequence of SEQ ID NO:6. In certain embodiments, polynucleotides comprises a nucleic acidsequence that is at least about 90% identical to the nucleic acidsequence of SEQ ID NO: 6. In certain embodiments, polynucleotidescomprises a nucleic acid sequence that is at least about 95% identicalto the nucleic acid sequence of SEQ ID NO: 6. In certain embodiments,polynucleotides comprises a nucleic acid sequence that is at least about97% identical to the nucleic acid sequence of SEQ ID NO: 6. In certainembodiments, polynucleotides comprises a nucleic acid sequence that isat least about 98% identical to the nucleic acid sequence of SEQ ID NO:6. In certain embodiments, polynucleotides comprises a nucleic acidsequence that is at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 97%, at least about 98%, or atleast about 99% identical to the nucleic acid sequence of SEQ ID NO: 6.

Properties of the Inventive Polypeptides

The inventive polypeptides provided herein are fluorescent with reducedion pumping activity compared to a natural member of the archaerhodopsinfamily of proteins from which it is derived. In certain embodiments, thepolypeptide of any one of the preceding claims, wherein the polypeptideis activated by contact with light having a non-blue light wavelength.In certain embodiments, the polypeptide is activated by contact with atleast one or all of yellow light, orange, or red light. In certainembodiments, the polypeptide is minimally activated or not at allactivated by contact with blue light. In certain embodiments, thepolypeptide is activated by contact with red light having a wavelengthof at least about 590 nm. In certain embodiments, the polypeptide isactivated by contact with red light having a wavelength of at leastabout 600 nm. In certain embodiments, the polypeptide is activated bycontact with red light having a wavelength of at least about 620 nm. Incertain embodiments, the polypeptide is activated by contact with redlight having a wavelength of at least about 630 nm. In certainembodiments, the polypeptide is activated by contact with red lighthaving a wavelength of at least about 640 nm. In certain embodiments,the polypeptide is activated by contact with red light having awavelength of at least about 650 nm. In certain embodiments, thepolypeptide is activated by contact with red light having a wavelengthof about 600 nm to about 700 nm. In certain embodiments, the polypeptideis activated by contact with red light having a wavelength of about 620nm to about 690 nm.

In certain embodiments, the polypeptide when contacted with blue light,the polypeptide is activated not at all, or at least less than 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12, 13%, 14%, 15%, 16%, 17%, 18%,19%, or 20% of the level of activation of the polypeptide when contactedwith red light.

In certain embodiments, the inventive polypeptide does not distort thewaveform of action potentials in various cells, for example, inmammalian neurons, skeletal myocytes, cardiac cells, glial cells,pancreatic beta cells, or in endothelial cell, for example in mammaliancardiomyocytes (e.g., human induced pluripotent stem cell (iPS)-derivedcardiomyocytes). In certain embodiments, the inventive polypeptide doesnot distort the waveform of action potentials of neurons. In certainembodiments, the inventive polypeptide does not distort the waveform ofaction potentials of cardiac cells. In certain embodiments, theinventive polypeptide does not distort the waveform of action potentialsof skeletal muscle cells. In certain embodiments, the inventivepolypeptide does not distort the waveform of action potentials that riseand fall in greater than or equal to about 0.05 ms, greater than orequal to about 0.1 ms, greater than or equal to about 1 ms, greater thanor equal to about 1.5 ms, greater than or equal to about 5 ms, greaterthan or equal to about 10 ms, or greater than or equal to about 12 ms.In certain embodiments, the inventive polypeptide does not distort thewaveform of action potentials that rise and fall in greater than orequal to about 0.05 ms. In certain embodiments, the inventivepolypeptide does not distort the waveform of action potentials that riseand fall in greater than or equal to about 0.1 ms. In certainembodiments, the inventive polypeptide does not distort the waveform ofaction potentials that rise and fall in greater than or equal to about 1ms. In certain embodiments, the inventive polypeptide does not distortthe waveform of action potentials that rise and fall in greater than orequal to about 1.5 ms. In certain embodiments, the inventive polypeptidedoes not distort the waveform of action potentials that rise and fall ingreater than or equal to about 5 ms. In certain embodiments, theinventive polypeptide does not distort the waveform of action potentialsthat rise and fall in about 0.05 ms to 1.5 ms, or about 1.5 ms to 5 ms,or about 5 ms to 12 ms. For example, the fluorescence of QuasAr1responds to a step in voltage in <0.05 ms, while QuasAr2 responds to astep in voltage in about 1.2 ms at room temperature. Thus QuasAr1 doesnot distort the waveform of action potentials that rise and fall in 0.1ms, such as those occurring in fast-spiking interneurons; and QuasAr2does not distort action potentials that occur in about 1 millisecond orlonger, such as cardiac action potentials.

In certain embodiments, the polypeptide shows a change in fluorescenceover the physiological voltage range of about −70 mV to about +30 mV. Incertain embodiments, the polypeptide shows a change in fluorescence overthe physiological voltage range of about −50 mV to about +30 mV. Incertain embodiments, the polypeptide shows a change in fluorescence overthe physiological voltage range of about −100 mV to about +50 mV. Incertain embodiments, the polypeptide shows a change in fluorescence overthe physiological voltage range of about −80 mV to about +50 mV. Incertain embodiments, the polypeptide shows a change in fluorescence overthe physiological voltage range of about −70 mV to about +30 mV. Incertain embodiments, the polypeptide shows a change in fluorescence overthe physiological voltage range of about −30 mV to about +30 mV. Incertain embodiments, the polypeptide shows a change in fluorescence overthe physiological voltage range of the subthreshold voltage dynamics inneurons. In certain embodiments, the polypeptide shows a change influorescence over the physiological voltage range of about −80 mV toabout −40 mV. In certain embodiments, the polypeptide shows a change influorescence over the physiological voltage range of inhibitory andexcitatory post-synaptic potentials. In certain embodiments, thepolypeptide shows a change in fluorescence over the physiologicalvoltage range of about −70 mV to about −50 mV. In certain embodiments,the change in flouorescence is large, e.g., at least about 20% per 100mV, at least about 30% per 100 mV, is at least about 40% per 100 mV, isat least about 50% per 100 mV, is at least about 60% per 100 mV, is atleast about 70% per 100 mV, is at least about 80% per 100 mV, or is atleast about 90% per 100 mV. In certain embodiments, the change inflouorescence is large and approximately linear.

In certain embodiments, the inventive polypeptide exhibits afluoresecent quantum yield of about 1×10⁻³ to about 30×10⁻³. In certainembodiments, the inventive polypeptide exhibits a fluoresecent quantumyield of about 1×10⁻³ to about 15×10⁻³. In certain embodiments, theinventive polypeptide exhibits a fluoresecent quantum yield of about15×10⁻³ to about 30×10⁻³. In certain embodiments, the inventivepolypeptide exhibits a fluoresecent quantum yield of about 3×10⁻³ toabout 5×10⁻³. In certain embodiments, the inventive polypeptide exhibitsa fluoresecent quantum yield of about 5×10⁻³ to about 7×10⁻³. In certainembodiments, the inventive polypeptide exhibits a fluoresecent quantumyield of about 7×10⁻³ to about 9×10⁻³. In certain embodiments, theinventive polypeptide exhibits a fluoresecent quantum yield of about4×10⁻³. In certain embodiments, the inventive polypeptide exhibit afluoresecent quantum yield of about 8×10⁻³.

In certain embodiments, the inventive polypeptide exhibits afluoresecent quantum yield enhanced by about 10-fold to about 20-foldcompared to Arch (D95N), which is described in US Patent Application No.2013/0224756, incorporated herein by reference in its entirety. Incertain embodiments, the inventive polypeptide exhibits a fluoresecentquantum yield enhanced by about 10-fold to about 15-fold compared toArch (D95N). In certain embodiments, the inventive polypeptide exhibitsa fluoresecent quantum yield enhanced by about 15-fold to about 20-foldcompared to Arch (D95N). In certain embodiments, the inventivepolypeptide exhibits a fluoresecent quantum yield enhanced by about10-fold. In certain embodiments, the inventive polypeptide exhibits afluoresecent quantum yield enhanced by about 19-fold.

In certain embodiments, the inventive polypeptide exhibits afluoresecent quantum yield enhanced by about 2-fold to about 20-foldcompared to wild-type Arch, when excited at at a wavelength of 640 nmand under an intensity of 500 mW/cm². In certain embodiments, theinventive polypeptide exhibits a fluoresecent quantum yield enhanced byabout 3-fold to about 17-fold compared to wild-type Arch. In certainembodiments, the inventive polypeptide exhibits a fluoresecent quantumyield enhanced by about 3-fold to about 15-fold compared to wild-typeArch. In certain embodiments, the inventive polypeptide exhibits afluoresecent quantum yield enhanced by about 2-fold to about 5-foldcompared to wild-type Arch. In certain embodiments, the inventivepolypeptide exhibits a fluoresecent quantum yield enhanced by about13-fold to about 17-fold compared to wild-type Arch. In certainembodiments, the inventive polypeptide exhibits a fluoresecent quantumyield enhanced by about 3-fold to about 4-fold compared to wild-typeArch. In certain embodiments, the inventive polypeptide exhibits afluoresecent quantum yield enhanced by about 15-fold compared towild-type Arch.

In certain embodiments, the inventive polypeptide exhibits afluoresecent quantum yield is enhanced by about 1.5-fold to about 5-foldcompared to wild-type Arch when excited at at a wavelength of 640 nm andunder an intensity of at least 100 W/cm². In certain embodiments, theinventive polypeptide exhibits a fluoresecent quantum yield enhanced byabout 2-fold to about 3-fold compared to wild-type Arch. In certainembodiments, the inventive polypeptide exhibits a fluoresecent quantumyield enhanced by about 2.5-fold compared to wild-type Arch.

In certain embodiments, the inventive polypeptide exhibits a brightnessthat is linear with the illumination intensity.

In certain embodiments, the inventive polypeptide exhibits a sensitivityof about 1.25-fold to 2.5-fold higher than the sensitivity of Arch orArch (D95N) between −100 mV and +50 mV. In certain embodiments, theinventive polypeptide exhibits a sensitivity of about 25% to about 100%per 100 mV. In certain embodiments, the inventive polypeptide exhibits asensitivity of about 25% to about 35% per 100 mV. In certainembodiments, the inventive polypeptide exhibits a sensitivity of about30% to about 40% per 100 mV. In certain embodiments, the inventivepolypeptide exhibits a sensitivity of about 35% to about 50% per 100 mV.In certain embodiments, the inventive polypeptide exhibits a sensitivityof about 50% to about 70% per 100 mV. In certain embodiments, theinventive polypeptide exhibits a sensitivity of about 70% to about 100%per 100 mV. In certain embodiments, the inventive polypeptide exhibits asensitivity of about 85% to about 95% per 100 mV. In certainembodiments, the inventive polypeptide exhibits a sensitivity of about90% per 100 mV.

In certain embodiments, the inventive polypeptide has a step responsetime constant of less than about 41 ms when measured at roomtemperature. In certain embodiments, the inventive polypeptide has astep response time constant of less than about 15 ms when measured atroom temperature. In certain embodiments, the inventive polypeptides hasa step response time constant of less than about 6 ms when measured atroom temperature. In certain embodiments, the inventive polypeptides hasa step response time constant of less than about 1.5 ms when measured atroom temperature. In certain embodiments, the inventive polypeptides hasa step response time constant of less than about 1 ms when measured atroom temperature. In certain embodiments, the inventive polypeptides hasa step response time constant of less than about 0.6 ms when measured atroom temperature. In certain embodiments, the inventive polypeptide hasa step response time constant of about 0.1 ms to about 15 ms whenmeasured at room temperature. In certain embodiments, the inventivepolypeptides has a step response time constant of about 0.05 ms to about0.6 ms when measured at room temperature. In certain embodiments, theinventive polypeptides has a step response time constant of about 0.3 mswhen measured at about 34° C. In certain embodiments, the inventivepolypeptides has a step response time constant that is mono-exponential.In certain embodiments, the inventive polypeptides has a step responsetime constant that is bi-exponential.

In certain embodiments, the inventive polypeptides has a photobleachingtime constant of about 400 s to about 1200 s. In certain embodiments,the inventive polypeptides has a photobleaching time constant of about400 s to about 500 s. In certain embodiments, the inventive polypeptideshas a photobleaching time constant of about 900 s to about 1100 s.

The inventive polypeptides also show far red excitation spectrum, whichmeans that the inventive polypeptides absorb wavelength in the red lightend of the spectrum. In certain embodiments, the inventive polypeptidescan be excited with light ranging from 600 nm to 690 nm light, and theemission is in the near infrared region, peaked at 750 nm. The emissionis farther to the red than any existing fluorescent protein. Thesewavelengths coincide with low cellular autofluorescence and goodtransmission through tissue. This feature makes these proteinsparticularly useful in optical measurements of action potential as thespectrum facilitates imaging with high signal-to-noise, as well asmulti-spectral imaging in combination with other fluorescent probes.

The GEVIs also exhibit high targetability. GEVIs can be imaged inprimary neuronal cultures, cardiomyocytes (HL-1 and human iPSC-derived),HEK cells, and Gram positive and Gram negative bacteria. In certainembodiments, the GEVIs have been targeted to the endoplasmic reticulumand mitochondria. The GEVIs can be used for in vivo imaging in C.elegans, zebrafish, and mice.

With the microbial rhodopsin constructs of the invention furthercomprising a cell type- and/or a time-specific promotors, one can imagemembrane potential in any optically accessible cell type or organelle ina living organism.

In certain embodiments, an inventive polypeptides comprises, consistsof, or consists essentially of at least three elements: a promoter, aninventive polypeptide, one or more targeting motifs, and an optionalsecond fluorescent protein.

In certain embodiment, at least one element from each group of promoter,voltage indicator, and targeting motif are selected to create an VIPwith the desired properties. A second polypeptide is optionally selectedto create a fusion protein with the voltage indicators provided herein.In some embodiments, methods and compositions for voltage sensing asdescribed herein involves selecting: 1) an archaerhodopsin protein orvariant thereof; 2) one or more mutations to imbue the protein withsensitivity to voltage or to other quantities of interest (e.g.,increased brightness) and to eliminate light-driven charge pumping; 3)codon usage appropriate to the host species; 4) a promoter and targetingsequences to express the protein in cell types of interest and to targetthe protein to the sub-cellular structure of interest; 5) an optionalfusion with a second fluorescent protein to provide ratiometric imaging;6) a chromophore comprising, e.g., retinal, dimethylamino retinal, or3,4 dehydro retinal, to optionally insert into the archaerhodopsinprotein or variant thereof; and 7) an optical imaging scheme.

Fusion Proteins and FRET Pairs

The inventive polypeptides are termed genetically encoded voltageindicators (GEVIs). Provide herein are GEVIs with improved brightness,that function through electrochromic fluorescence resonance energytransfer (eFRET) between an appended fluorescent protein and theArchaerhodopsin-based chromophore, retinal (i.e., a FRET pair).

These eFRET-based GEVIs have enhanced brightness and comparable speedrelative to the direct fluorescence of the individual GEVIs. In eFRET,the electronic shifts of an acceptor polypeptide can be used to alterthe degree of spectral overlap between the emission of the donorpolypeptide and the absorption of the acceptor polypeptide, therebyaltering the degree of nonradiative quenching of an acceptor polypeptideby the donor polypeptide. For example, the more overlap between thedonor emission spectrum with the acceptor absorption spectrum means ahigher efficiency of donor fluorescence quenching by the acceptor. Theless overlap between the donor emission spectrum with the acceptorabsorption spectrum means a lower efficiency of donor fluorescencequenching by the acceptor. FIG. 6 generally illustrates eFRET-basedvoltage indicators. In the FRET pairs provided herein, the GEVIs are theelectrochromic quencher, which exhibit changes in absorbance in responseto changes in membrane voltage due (voltage-dependent absorption).Voltage-dependent changes in the absorption spectrum of the GEVI'sretinal chromophore lead to voltage-dependent rates of nonradiative FRETbetween a fluorescent protein and the retinal. Retinal in its absorbing,fluorescent (protonated) state quenches the GFP, while retinal in thenon-absorbing, non-fluorescent state does not quench the GFP. Thefluorescence of a donor polypeptide would be decreased or increaseddepending on how weakly or strongly the acceptor polypeptide absorbs thefluorescence of a donor polypeptide, which is dependent on the spectraloverlap. It has been observed that membrane voltage changes thefluorescence of the GEVIs provided herein. In view of this observation,the fluorescence changes of the GEVIs are a result of changes in itsabsorbance spectrum, and therefore, the GEVIs are useful forvoltage-dependent quenching of a second fluorescent protein appended toa GEVI.

Provided herein are fusion proteins comprising the inventivepolypeptides described herein (i.e., the GEVIs). In certain embodiments,the fusion proteins comprises the inventive polypeptide or variantthereof provided herein and a second polypeptide. In certainembodiments, the second polypeptide is a fluorescent polypeptide orhomologues thereof. In certain embodiments, the fusion proteins form anelectrochromic FRET pair (i.e., spectral shift FRET (ssFRET)). Thefluorescence of the second polypeptide is blue-shifted compared to theabsorbance of the polypeptide or variant thereof provided herein. Incertain embodiments, the fluorescence of the second polypeptide iswithin the orange light, yellow light, green light, blue light, indigolight, or violet light region of the visible spectrum.

The fusion proteins provided herein have enhanced brightness and can beused in 2-photon imaging, ratiometric voltage imaging, and multimodalsensors for simultaneous measurement of voltage and concentration. Thefusion proteins provided herein can be used in any of the methods of thepresent invention. The fusion proteins provided herein can be preparedusing a nucleic acid encoding the inventive polypeptides that isoperably linked to or fused with an additional fluorescent protein. Incertain embodiments, the second fluorescent protein is GFP, YFP,citrine, mOrange2, mKate2, mRuby2, or a variant thereof. In certainembodiments, the fusion proteins can be covalently joined together. Incertain embodiments, the fusion proteins can be non-covalently joinedtogether. In certain embodiments, the fusion proteins are joinedtogether using standard protein linkers.

In certain embodiments, the fusion proteins comprise an amino acidsequence of SEQ ID NO: 2, and an amino acid sequence of a secondfluorescent protein. In certain embodiments, the fusion proteincomprises a variant of an amino acid sequence of SEQ ID NO: 2, and anamino acid sequence of a second fluorescent protein. In certainembodiments, the second fluorescent protein is GFP, YFP, citrine,mOrange2, mKate2, mRuby2, or a variant thereof. In certain embodiments,the second fluorescent protein has an emission wavelength range that isblue-shifted compared to the GEVIs. In certain embodiments, the fusionproteins comprise an amino acid sequence of SEQ ID NO: 2, or a variantthereof, and an amino acid sequence of GFP, or a variant thereof. Incertain embodiments, the fusion proteins comprise an amino acid sequenceof SEQ ID NO: 2, or a variant thereof, and an amino acid sequence ofYFP, or a variant thereof. In certain embodiments, the fusion proteinscomprise an amino acid sequence of SEQ ID NO: 2, or a variant thereof,and an amino acid sequence of citrine, or a variant thereof. In certainembodiments, the fusion proteins comprise an amino acid sequence of SEQID NO: 2, or a variant thereof, and an amino acid sequence of mOrange2,or a variant thereof. In certain embodiments, the fusion proteinscomprise an amino acid sequence of SEQ ID NO: 2, or a variant thereof,and an amino acid sequence of mKate2, or a variant thereof. In certainembodiments, the fusion proteins comprise an amino acid sequence of SEQID NO: 2, or a variant thereof, and an amino acid sequence of mRuby2, ora variant thereof.

In certain embodiments, the fusion proteins comprise an amino acidsequence of SEQ ID NO: 3 and an amino acid sequence of a secondfluorescent protein. In certain embodiments, the fusion proteincomprises a variant of an amino acid sequence of SEQ ID NO: 3. Incertain embodiments, the second fluorescent protein is GFP, YFP,citrine, mOrange2, mKate2, mRuby2, or a variant thereof. In certainembodiments, the second fluorescent protein has an emission wavelengthrange that is blue-shifted compared to the GEVIs. In certainembodiments, the fusion proteins comprise an amino acid sequence of SEQID NO: 3, or a variant thereof, and an amino acid sequence of GFP, or avariant thereof. In certain embodiments, the fusion proteins comprise anamino acid sequence of SEQ ID NO: 3, or a variant thereof, and an aminoacid sequence of YFP, or a variant thereof. In certain embodiments, thefusion proteins comprise an amino acid sequence of SEQ ID NO: 3, or avariant thereof, and an amino acid sequence of citrine, or a variantthereof. In certain embodiments, the fusion proteins comprise an aminoacid sequence of SEQ ID NO: 3, or a variant thereof, and an amino acidsequence of mOrange2, or a variant thereof. In certain embodiments, thefusion proteins comprise an amino acid sequence of SEQ ID NO: 3, or avariant thereof, and an amino acid sequence of mKate2, or a variantthereof. In certain embodiments, the fusion proteins comprise an aminoacid sequence of SEQ ID NO: 3, or a variant thereof, and an amino acidsequence of mRuby2, or a variant thereof.

In certain embodiments, the fusion proteins comprise an amino acidsequence having at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% sequence homology to SEQ ID NO:2, and an amino acid sequence of a second fluorescent protein. Incertain embodiments, the fusion proteins comprise an amino acid sequencehaving at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 97%, at least about 98%, or at leastabout 99% sequence homology to SEQ ID NO: 3, and an amino acid sequenceof a second fluorescent protein.

In certain embodiments, the fusion proteins comprise an amino acidsequence having at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% sequence identity to SEQ ID NO:2, and an amino acid sequence of a second fluorescent protein. Incertain embodiments, the fusion proteins comprise an amino acid sequencehaving at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% sequence identity to SEQ ID NO: 3, and anamino acid sequence of a second fluorescent protein.

It is useful to have fusion proteins wherein the fused proteins span thevisible spectrum. In certain embodiments, the second fluorescent proteinhas an emission wavelength range that is blue-shifted compared to theGEVIs. In certain embodiments, the second fluorescent protein is GFP,YFP, Citrine, mOrange2, mKate2, mRuby2, or a variant thereof. In certainembodiments, the second fluorescent protein is GFP or a variant thereof.In certain embodiments, the second fluorescent protein is YFP or avariant thereof. In certain embodiments, the second fluorescent proteinis citrine or a variant thereof. In certain embodiments, the secondfluorescent protein is mOrange2 or a variant thereof. In certainembodiments, the second fluorescent protein is mKate2 or a variantthereof. In certain embodiments, the second fluorescent protein ismRuby2 or a variant thereof.

In certain embodiments, the second fluorescent protein is fused to theGEVIs at either the N-terminus or the C-terminus of the GEVI. In certainembodiments, the second fluorescent protein is fused to the GEVIs at theC-terminus of the GEVI. In certain embodiments, the fusion proteincomprises a polypeptide comprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 3 fused at its C-terminus to a second fluorescentprotein. In certain embodiments, the two polypeptides of the fusionprotein are linked via a short linker. In certain embodiments, the shortlinker comprises 2 to 5 amino acids. In certain embodiments, the shortlinker comprises the amino acids Leu and Arg. In certain embodiments,the short linker comprises 2 or 3 amino acids. In certain embodiments,the short linker comprises 2 amino acids. In certain embodiments, theshort linker is Leu and Arg.

In certain embodiments, the fusion protein comprises a polypeptidecomprising an amino acid sequence of SEQ ID NO: 2 fused at itsC-terminus to a second fluorescent protein. In certain embodiments, thefusion protein comprises a polypeptide comprising an amino acid sequenceof SEQ ID NO: 2 fused at its C-terminus to a second fluorescent proteinselected from GFP, YFP, citrine, mOrange2, mKate2, mRuby2 and variantsthereof. In certain embodiments, the fusion protein comprises apolypeptide comprising an amino acid sequence of SEQ ID NO: 2 fused atits C-terminus to a GFP or a variant thereof. In certain embodiments,the fusion protein comprises a polypeptide comprising an amino acidsequence of SEQ ID NO: 2 fused at its C-terminus to a YFP or a variantthereof. In certain embodiments, the fusion protein comprises apolypeptide comprising an amino acid sequence of SEQ ID NO: 2 fused atits C-terminus to citrine or a variant thereof. In certain embodiments,the fusion protein comprises a polypeptide comprising an amino acidsequence of SEQ ID NO: 2 fused at its C-terminus to a mOrange2 or avariant thereof. In certain embodiments, the fusion protein comprises apolypeptide comprising an amino acid sequence of SEQ ID NO: 2 fused atits C-terminus to mKate2 or a variant thereof. In certain embodiments,the fusion protein comprises a polypeptide comprising an amino acidsequence of SEQ ID NO: 2 fused at its C-terminus to mRuby2 or a variantthereof.

In certain embodiments, the fusion protein comprises a polypeptidecomprising an amino acid sequence of SEQ ID NO: 3 fused at itsC-terminus to a second fluorescent protein. In certain embodiments, thefusion protein comprises a polypeptide comprising an amino acid sequenceof SEQ ID NO: 3 fused at its C-terminus to a second fluorescent proteinselected from GFP, YFP, citrine, mOrange2, mKate2, mRuby2 and variantsthereof. In certain embodiments, the fusion protein comprises apolypeptide comprising an amino acid sequence of SEQ ID NO: 3 fused atits C-terminus to a GFP or a variant thereof. In certain embodiments,the fusion protein comprises a polypeptide comprising an amino acidsequence of SEQ ID NO: 3 fused at its C-terminus to a YFP or a variantthereof. In certain embodiments, the fusion protein comprises apolypeptide comprising an amino acid sequence of SEQ ID NO: 3 fused atits C-terminus to a citrine or a variant thereof. In certainembodiments, the fusion protein comprises a polypeptide comprising anamino acid sequence of SEQ ID NO: 3 fused at its C-terminus to amOrange2 or a variant thereof. In certain embodiments, the fusionprotein comprises a polypeptide comprising an amino acid sequence of SEQID NO: 3 fused at its C-terminus to a mKate2 or a variant thereof. Incertain embodiments, the fusion protein comprises a polypeptidecomprising an amino acid sequence of SEQ ID NO: 3 fused at itsC-terminus to a mRub2 or a variant thereof.

Since the GEVI fusion proteins (i.e., eFRET GEVIs) span the visiblespectrum, the GEVI fusion proteins provided herein are useful inmulticolor voltage imaging. The high brightness of the eFRET GEVIs alsomakes these fusion proteins useful for voltage imaging in vivo. The highbrightness of the eFRET GEVIs also makes these proteins useful forvoltage imaging with two-photon excitation. Accordingly, provided is amethod of detecting action potentials in various cells, for example, inmammalian neurons, skeletal myocytes, cardiac cells, glial cells,pancreatic beta cells, or in endothelial cell, for example in mammaliancardiomyocytes (e.g., human induced pluripotent stem cell (iPS)-derivedcardiomyocytes). Cardiomyocytes include ventricular, atrial, and nodalcells. Such methods comprises transfecting neurons or cardiac cells withthe GEVI fusion proteins. In certain embodiments, the fusion proteintrafficks to sub-cellular compartments. In certain embodiments, thefusion protein trafficks to the endoplasmic reticulum. In certainembodiments, the fusion protein localizes to a membrane of the cell. Incertain embodiments, the fusion protein localizes to the plasmamembrane. In certain embodiments, the fusion protein localizes to themembrane of sub-cellular compartments.

In certain embodiments, the fusion proteins enables ratiometricdetermination of membrane potential. Since it has been observed thatrate of eFRET decreases with increasing distance between thepolypeptides, such embodiments employ the use of a long linker betweenthe two polypeptides being fused such that the polypeptides do not undergo eFRET. Since the fluorescence of the second fluorescent protein isindependent of membrane potential, the ratio of fluorescence for theinventive polypeptides to the fluorescene of the second fluorescentprotein provides a measure of membrane potential that is independent ofvariations in expression level, illumination, or movement.

Membrane potential is only one of several mechanisms of signaling withincells. In certain applications, it is desirable to correlate changes inmembrane potential with changes in concentration of other species, suchas Ca²⁺, H⁺ (i.e., pH), Na⁺, K⁺, Cl⁻, ATP, and cAMP. The GEVIs providedherein can also be useful in multimodal sensor applications where thevisible spectrum is used for other imaging modalities such assimultaneously measuring the concentrations of these other ions.Examples of other fusion proteins include the GEVIs provided hereinfused with a fluorescent pH indicator (e.g., pHluorin) or with afluorescent Ca²⁺ indicator (e.g., GCaMP6). In such applications, thesecond fluorescent polypeptide would interfere with such applications.However, the removal of the second fluorescent polypeptide may interferewith, for example, trafficking properties of the GEVIs. Thus, the secondfluorescent polypeptide of the fusion protein can be modified to thecorresponding non-fluorescent variant. Accordingly, in certainembodiments, the fusion proteins comprise an amino acid sequence of SEQID NO: 2 and a second fluorescent polypeptide, wherein secondfluorescent polypeptide is modified to the corresponding non-fluorescentvariant. In certain embodiments, the fusion proteins comprise an aminoacid sequence of SEQ ID NO: 3 and a second fluorescent polypeptide,wherein second fluorescent polypeptide is modified to the correspondingnon-fluorescent variant. In certain embodiments, the second fluorescentpolypeptide is mOrange2. In certain embodiments, the mOrange2polypeptide is mutated to the non-fluorescent form. In certainembodiments, the mOrange2 polypeptide mutant comprises a Y72A mutation.The protein ID number of mOrange is D0VWW2.

Additional second fluorescent proteins include Venus, EGFP, EYFP, EBFB,DsRed, RFP, and fluorescent variants thereof.

Nucleic Acid Constructs and Expression Vectors

Provided herein are nucleic acid constructs comprising the inventivepolynucleotides. In certain embodiments, the nucleic acid constructcomprises a polynucleotide encoding an amino acid sequence of SEQ ID NO:1, wherein at least one of the amino acids at positions 60, 80, 95, 106,or 161 have been mutated. In certain embodiments, the nucleic acidconstruct comprises a polynucleotide encoding an amino acid sequence ofSEQ ID NO: 1, wherein the amino acids at positions 60, 80, 95, 106, and161 have been mutated. In certain embodiments, the nucleic acidconstruct comprises a polynucleotide encoding an amino acid sequence ofSEQ ID NO: 2. In certain embodiments, the nucleic acid constructcomprises a polynucleotide encoding an amino acid sequence of SEQ ID NO:3.

In certain embodiments, the nucleic acid construct comprises apolynucleotide of SEQ ID NO: 4, wherein the polynucleotide encodes forat least one amino acid mutation at positions 60, 80, 95, 106, or 161 ofthe encoded polypeptide. In certain embodiments, the nucleic acidconstruct comprises a polynucleotide of SEQ ID NO: 4, wherein thepolynucleotide encodes for amino acid mutation at positions 60, 80, 95,106, and 161 of the encoded polypeptide. In certain embodiments, thenucleic acid construct comprises a polynucleotide of SEQ ID NO: 5. Incertain embodiments, the nucleic acid construct comprises apolynucleotide of SEQ ID NO: 6.

In certain embodiments, the nucleic acid construct comprises a secondpolynucleotide encoding a second polypeptide as described herein. Incertain embodiments, the second polypeptide is fluorescent. In certainembodiments, the second fluorescent polypeptide is GFP, YFP, citrine,mOrange2, mKate2, mRuby2, or a variant thereof. In certain embodiments,the second fluorescent polypeptide is capable of indicating ionconcentration. In certain embodiments, the ion concentration indicatedis calcium or pH. In certain embodiments, the two polypeptides areconnected to encode a fusion protein comprising the inventivepolypeptides and a second fluorescent protein.

In certain embodiments, the nucleic acid construct comprises a promotersequence to control the expression of the polynucleotide orpolynucleotides. The promoter sequence is operatively linked to thepolynucleotide sequence that encode the inventive GEVIs. In certainembodiments, the nucleic acid construct comprises a pan cellularpromoter. In certain embodiments, the pan cellular promoter is a CAGenhancer, CMB, or ubiquitin, as described in US Patent Publication No.2013/0224756, incorporated by reference. In certain embodiments, thenucleic acid construct comprises a neuron specific promoter sequence. Incertain embodiments, the neuron specific promoter isCa²⁺-calmodulin-dependent protein kinase II (CaMKIIα) promoter. Incertain embodiments, the nucleic acid construct comprises a promoterthat is a human synapsin (hSyn) promoter. In certain embodiments, thenucleic acid construct comprises a promoter that is a GAD67 promoter.

In certain embodiments, the nucleic acid construct comprises a firstpromoter sequence to control the expression of the inventivepolynucleotides described herein, and a second promoter sequence tocontrol the expression of the second polynucleotides described herein,said first promoter sequence and said second promoter sequence aredifferent from each other. In certain embodiments, the secondpolynucleotides encode the fluorescent polypeptides or thenon-fluorescent version such as GFP, YFP, citrine, mOrange2, mKate2,mRuby2, or a variant thereof.

Further provided herein are expression vectors comprising any of theaforementioned inventive polynucleotides or the nucleic acid constructs.The term “vector” refers to a carrier DNA molecule into which a nucleicacid sequence can be inserted for introduction into a host cell. Vectorsuseful in the methods provided may include additional sequencesincluding, but not limited to one or more signal sequences and/orpromoter sequences, or a combination thereof. An “expression vector” isa specialized vector that contains the necessary regulatory regionsneeded for expression of a gene of interest in a host cell such astranscription control elements (e.g. promoters, enhancers, andtermination elements). Expression vectors and methods of their use arewell known in the art. Non-limiting examples of suitable expressionvectors and methods for their use are provided herein. Nucleic acidconstructs may be integrated and packaged into non-replicating,defective viral genomes like Adenovirus, Adeno-associated virus (AAV),or Herpes simplex virus (HSV) or others, including retroviral andlentiviral vectors, for infection or transduction into cells.

In certain embodiments, the vector comprises a trafficking sequence. Theinventive polypeptides described herein can be targeted to intracellularorganelles, including mitochondria, the endoplasmic reticulum, thesarcoplasmic reticulum, synaptic vesicles, and phagosomes. In certainembodiments, the vector comprises a membrane-targeting nucleic acidsequence operatively linked to to the polynucleotide encoding theinventive polypeptide. In certain embodiments, the membrane-targetingnucleic acid is a plasma membrane targeting nucleic acid sequence. Incertain embodiments, the membrane-targeting nucleic acid sequence is asubcellular compartment-targeting nucleic acid sequence. In certainembodiments, the subcellular compartment is selected from amitochondrial membrane, an endoplasmic reticulum, a sarcoplasticreticulum, a nuclear membrane, a synaptic vesicle, an endosome, and aphagosome. In certain embodiments, the subcellular compartment is theendoplasmic reticulum, the mitochondrial inner membrane, the nuclearmembrane, or a synaptic vesicle. In certain embodiments, the inventivepolypeptides described herein can be targeted to membrane regions suchas the plasma membrane or to membranes of sub-cellular compartments.

In certain embodiments, the vector also includes one, two, or morenucleic acid signal sequences operatively linked to the polynucleotidesequence encoding the inventive polypeptides. In certain embodiments,the vector is a plasmid vector, cosmid vector, viral vector, or anartificial chromosome.

In certain embodiments, the vector is a lentiviral vector. In certainembodiments, the nucleic acid constructs comprising the inventivepolynucleotides are incorporated into a lentiviral vector under theCaMKIIα promoter, adapted from Addgene plasmid 22217.

A vector can also further comprises at least one of the following: amarker gene, a reporter gene, an antibiotic-resistance gene, an enhancersequence, a gene encoding a selected gene product, a polyadenylationsite, and a regulatory sequence.

Vectors useful in methods of the invention may include additionalsequences including, but not limited to, one or more signal sequencesand/or promoter sequences, or a combination thereof. Expression vectorsand methods of their use are well known in the art. Non-limitingexamples of suitable expression vectors and methods for their use areprovided herein.

In certain embodiments of the invention, a vector may be a lentiviruscomprising the gene for a light-activated ion channel polypeptide of theinvention, such as ChR64, ChR86, or a variant thereof. A lentivirus is anon-limiting example of a vector that may be used to create stable cellline.

Promoters that may be used in methods and vectors of the inventioninclude, but are not limited to, cell-specific promoters or generalpromoters. Methods for selecting and using cell-specific promoters andgeneral promoters are well known in the art. A non-limiting example of ageneral purpose promoter that allows expression of a light-activated ionchannel polypeptide in a wide variety of cell types—thus a promoter fora gene that is widely expressed in a variety of cell types, for example,a “housekeeping gene” can be used to express a light-activated ionchannel polypeptide in a variety of cell types. Non-limiting examples ofgeneral promoters are provided elsewhere herein and suitable alternativepromoters are well known in the art.

In certain embodiment, the inventive polypeptides are encoded by adelivery vector. Non-liminting exemplary vectors include: plasmids (e.g.pBADTOPO, pCI-Neo, pcDNA3.0), cosmids, and viruses (such as alentivirus, an adeno-associated virus, or a baculovirus). In certainembodiments, the vectors are bicistronic vectors for co-expression ofthe inventive polypeptides and another fluorescent protein. In certainembodiments, the separate vectors are used for the separate expressionof the inventive polypeptides and another fluorescent protein.

In certain embodiments, to express a fusion protein such asArch-mOrange2 variants in HeLa cells, the polynucleotide in the pBADvector was first amplified by PCR using primers Fw_BamHI_Kozak_Arch andRV_FP_ERex_stp_XbaI. This reverse primer encodes the endoplasmicreticulum (ER) export sequence from the inward-rectifier potassiumchannel Kir2.1 (FCYENE) (SEQ ID NO: 8), which has been reported to beeffective for improving the membrane trafficking of Arch in mammaliancells. In certain embodiments, the purified polynucleotide DNA wasdigested with BamHI and XbaI restriction enzymes and ligated into apurified plasmid, such as the pcDNA3.1 plasmid, that had been digestedwith the same two enzymes. The ligation reaction was used for thetransformation of electrocompetent E. coli strain, such as DH10B cells.Cells were plated, individual colonies were picked and grown, followedby a small-scale isolation of plasmid DNA. Each gene in the plasmid wasfully sequenced using T7_FW and BGH_RV primers. Plasmid DNA was thenused for subsequent cell transfection. In certain embodiments, the cellsbeing transfected are HeLa cells.

In certain embodiments, the vector used is a lentivirus vector. Toenable more accurate electrophysiological characterization via patchclamp in HEK cells and primary neuron cultures, the inventivepolynucleotides can be cloned into restriction enzyme sites, such as theBamHI/EcoRI sites, of a lentivirus vector such as FCK-Arch-GFP (Addgene:22217). This vector contains a CaMKIIα promoter and a WoodchuckHepatitis Virus Posttranscriptional Regulatory Element (WPRE) after the3′ end of the open reading frame. The Arch cDNA was generated by PCRusing forward primer FW_BamHI_Kozak_Arch_ValSer and overlapping reverseprimers RV_FP_TS and RV_TS_ERex_stp_EcoRI. These reverse primersintroduce a trafficking signal (TS) motif and endoplasmic reticulum (ER)export signal peptide sequence at the C-terminus of the inventivepolypeptide.

Table 4 summarizes exemplary embodiments that can be used to createviral constructs that express the genetically encoded voltage indicatorsprovided herein. The sequence listings can be found in US PatentApplication No. 2013/0224756, which is herein incorporated by referencein its entirety.

TABLE 4 Exemplary embodiments that can be used to gene constructs withthe inventive polypeptides. Virus backbone Lentivirus Promoter CamKII(neuron specific) CAG enhancer (pan cellular) CMV (pan cellular)Ubiquitin (pan cellular) Voltage-sensing domain Inventive polypeptidesprovided herein

An “inducible promoter” is a promoter that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to a “regulatory agent” (e.g., doxycycline), or a“stimulus” (e.g., heat). In the absence of a “regulatory agent” or“stimulus”, the DNA sequences or genes will not be substantiallytranscribed. The term “not substantially transcribed” or “notsubstantially expressed” means that the level of transcription is atleast 100-fold lower than the level of transcription observed in thepresence of an appropriate stimulus or regulatory agent; generally atleast 200-fold, 300-fold, 400-fold, 500-fold or more. As used herein,the terms “stimulus” and/or “regulatory agent” refers to a chemicalagent, such as a metabolite, a small molecule, or a physiological stressdirectly imposed upon the organism such as cold, heat, toxins, orthrough the action of a pathogen or disease agent. A recombinant cellcontaining an inducible promoter may be exposed to a regulatory agent orstimulus by externally applying the agent or stimulus to the cell ororganism by exposure to the appropriate environmental condition or theoperative pathogen. Inducible promoters initiate transcription only inthe presence of a regulatory agent or stimulus. Examples of induciblepromoters include the tetracycline response element and promotersderived from the β-interferon gene, heat shock gene, metallothioneingene or any obtainable from steroid hormone-responsive genes. Induciblepromoters which may be used in performing the methods of the presentinvention include those regulated by hormones and hormone analogs suchas progesterone, ecdysone and glucocorticoids as well as promoters whichare regulated by tetracycline, heat shock, heavy metal ions, interferon,and lactose operon activating compounds. For review of these systems seeGingrich and Roder, 1998, Annu Rev Neurosci 21, 377-405. Tissue specificexpression has been well characterized in the field of gene expressionand tissue specific and inducible promoters are well known in the art.These promoters are used to regulate the expression of the foreign geneafter it has been introduced into the target cell.

The promoter sequence may be a “cell-type specific promoter” or a“tissue-specific promoter” which means a nucleic acid sequence thatserves as a promoter, i.e., regulates expression of a selected nucleicacid sequence operably linked to the promoter, and which affectsexpression of the selected nucleic acid sequence in specific cells ortissues where membrane potential is desired to be measured. In someembodiments, the cell-type specific promoter is a leaky cell-typespecific promoter. The term “leaky” promoter refers to a promoter whichregulates expression of a selected nucleic acid primarily in one celltype, but cause expression in other cells as well. For expression of anexogenous gene specifically in neuronal cells, a neuron-specific enolasepromoter can be used (see Forss-Petter et al., 1990, Neuron 5: 187-197).For expression of an exogenous gene in dopaminergic neurons, a tyrosinehydroxylase promoter can be used. For expression in pituitary cells, apituitary-specific promoter such as POMC may be used (Hammer et al.,1990, Mol. Endocrinol. 4:1689-97). Examples of muscle specific promotersinclude, for example α-myosin heavy chain promoter, and the MCKpromoter. Other cell specific promoters active in mammalian cells arealso contemplated herein. Such promoters provide a convenient means forcontrolling expression of the exogenous gene in a cell of a cell cultureor within a mammal.

In some embodiments, the expression vector is a lentiviral vector.Lentiviral vectors useful for the methods and compositions describedherein can comprise a eukaryotic promoter. The promoter can be anyinducible promoter, including synthetic promoters, that can function asa promoter in a eukaryotic cell. For example, the eukaryotic promotercan be, but is not limited to, ecdysone inducible promoters, E1ainducible promoters, tetracycline inducible promoters etc., as are wellknown in the art. In addition, the lentiviral vectors used herein canfurther comprise a selectable marker, which can comprise a promoter anda coding sequence for a selectable trait. Nucleotide sequences encodingselectable markers are well known in the art, and include those thatencode gene products conferring resistance to antibiotics oranti-metabolites, or that supply an auxotrophic requirement. Examples ofsuch sequences include, but are not limited to, those that encodethymidine kinase activity, or resistance to methotrexate, ampicillin,kanamycin, chloramphenicol, puromycinor zeocin, among many others.

In some embodiments the viral vector is an adeno-associated virus (AAV)vector. AAV can infect both dividing and non-dividing cells and mayincorporate its genome into that of the host cell.

The type of vector one selects will also depend on whether theexpression is intended to be stable or transient.

The invention also provides cells that are genetically engineered toexpress the microbial rhodopsin VIPs. The cell may be engineered toexpress the VIP transiently or stably.

The invention provides methods of making both transiently expressingcells and cells and cell lines that express the microbial rhodopsinsstably.

Transient Expression.

One of ordinary skill in the art is well equipped to engineer cells thatare transiently transfected to express the VIPs or PROPS as describedherein. Transduction and transformation methods for transient expressionof nucleic acids are well known to one skilled in the art.

Transient transfection can be carried out, e.g., using calciumphosphate, by electroporation, or by mixing a cationic lipid with thematerial to produce liposomes, cationic polymers or highly branchedorganic compounds. All these are in routine use in genetic engineering.

One of ordinary skill in the art is well equipped to engineer cells thatstably express the VIPs or PROPS as described herein. These methods arealso in routine use in genetic engineering. Exemplary protocols can befound, e.g., in Essential Stem Cell Methods, edited by Lanza andKlimanskaya, published in 2008, Academic Press. For example, one cangenerate a virus that integrates into the genome and comprises aselectable marker, and infect the cells with the virus and screen forcells that express the marker, which cells are the ones that haveincorporated the virus into their genome. For example, one can generatea VSV-g psuedotyped lenti virus with a puromycin selectable marker inHEK cells according to established procedures. Generally, one can use astem cell specific promoter to encode a GFP if FACS sorting isnecessary. The hiPS cultures are cultivated on embryonic fibroblast (EF)feeder layers or on Matrigel in fibroblast growth factor supplemented EFconditioned medium. The cells are dissociated by trypsinization to asingle cell suspension The cells can be plated, e.g., 1×10⁵ cells on atissue culture 6-well plate pretreated with, e.g., Matrigel. To maintainthe cells in an undifferentiated state, one can use, e.g., EFconditioned medium. About 6 hours after plating, one can add virussupernatant to adhered cells (use 5×10⁶ IU virus per 1×10⁵ cells). Add 6μg/mL protamine sulfate to enhance virus infection. Cells are culturedwith the virus for 24 hours; washed, typically with PBS, and fresh mediais added with a selection marker, such as 1 μg/mL puromycin. The mediumis replaced about every 2 days with additional puromycin. Cellssurviving after 1 week are re-plated, e.g., using the hanging dropmethod to form EBs with stable incorporation of gene.

In some embodiments, it is advantageous to express a VIP (e.g., Arch 3D95N) in only a single cell-type within an organism, and further, ifdesired, to direct the sensor to a particular subcellular structurewithin the cell. Upstream promoters control when and where the gene isexpressed. Constructs are made that optimize expression in alleukaryotic cells. In one embodiment, the VIP is under the control of aneuron-specific promoter.

The promoter sequence can be selected to restrict expression of theprotein to a specific class of cells and environmental conditions.Common promoter sequences include, but are not limited to, CMV(cytomegalovirus promoter; a universal promoter for mammalian cells),14×UAS-E1b (in combination with the transactivator Gal4, this promoterallows combinatorial control of transgene expression in a wide array ofeukaryotes. Tissue-specific expression can be achieved by placing Gal4under an appropriate promoter, and then using Gal4 to drive theUAS-controlled transgene), HuC (drives pan-neuronal expression inzebrafish and other teleosts), ara (allows regulation of expression witharabinose in bacteria) and lac (allows regulation of expression withIPTG in bacteria).

In some embodiments, the VIP further comprises a localization ortargeting sequence to direct or sort the sensor to a particular face ofa biological membrane or subcellular organelle. Useful localizationsequences provide for highly specific localization of the protein, withminimal accumulation in other subcellular compartments. Examplelocalization sequences that direct proteins to specific subcellularstructures are provided in US Patent Publication No. 2013/0224756(incorporated by reference) and include nuclear (import signal),endoplasmic reticulum (import signal), endoplasmic reticulum (retentionsignal), peroxisome (import signal), peroxisome (import signal),mitochondrial inner membrane, mitochondrial outer membrane, plasmamembrane (cytosolic face), plasma membrane (cytosolic face),mitochondrial targeting sequence: human PINK1, mitochondrial targetingsequence: human serine protease HTRA2, mitochondrial targeting sequence:human cytochrome oxidase 1, mitochondrial targeting sequence: humancytochrome oxidase 2, mitochondrial targeting sequence: human proteinphospatase 1K, mitochondrial targeting sequence: human ATP synthasealpha, and mitochondrial targeting sequence: human frataxin.

Other examples of localization signals are described in, e.g., “ProteinTargeting”, chapter 35 of Stryer, L., Biochemistry (4th ed.). W. H.Freeman, 1995 and Chapter 12 (pages 551-598) of Molecular Biology of theCell, Alberts et al. third edition, (1994) Garland Publishing Inc. Insome embodiments, more than one discrete localization motif is used toprovide for correct sorting by the cellular machinery. For example,correct sorting of proteins to the extracellular face of the plasmamembrane can be achieved using an N-terminal signal sequence and aC-terminal GPI anchor or transmembrane domain.

Typically, localization sequences can be located almost anywhere in theamino acid sequence of the protein. In some cases the localizationsequence can be split into two blocks separated from each other by avariable number of amino acids. The creation of such constructs viastandard recombinant DNA approaches is well known in the art, as forexample described in Maniatis et al., Molecular Cloning A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y, 1989).

Targeting to the Plasma Membrane:

In some embodiments, constructs are designed to include signalingsequences to optimize localization of the protein to the plasmamembrane. These can include e.g., a C-terminal signaling sequence fromthe nicotinic acetylcholine receptor, and/or an endoplasmic reticulumexport motif from Kir2.1 comprising the sequence FCYENE (SEQ ID NO: 8).Examples of targeting sequences are provided in US Patent PublicationNo. 2013/0224756, incorporated herein by reference.

Additional improvements in plasma localization can be obtained by addingGolgi export sequences (e.g., from Kir2.1) and membrane localizationsequences (e.g., from Kir2.1) (Gradinaru et al. Cell (2010)). In someembodiments, the targeting sequence is selected to regulateintracellular transport of the protein to the desired subcellularstructure. In one embodiment the protein is targeted to the plasmamembrane of a eukaryotic cell. In this case the targeting sequence canbe designed following the strategy outlined in, e.g., Gradinaru et al.,“Molecular and Cellular Approaches for Diversifying and ExtendingOptogenetics,” Cell 141, 154-165 (2010). The term “signal sequence”refers to N-terminal domains that target proteins into a subcellularlocale e.g., the endoplasmic reticulum (ER), and thus are on their wayto the plasma membrane. Signal sequences used in optogenetic voltagesensors can be derived from the proteins 132-n-acetylcholine receptor(SS B2nAChR) and PPL. In addition, there is an endogenous signalingsequence on microbial rhodopsin proteins that can be harnessed forappropriate subcellular targeting. A trafficking signal (TS) canoptionally be inserted into the genome C-terminal to the microbialrhodopsin and N-terminal to the accessory fluorescent protein. In oneembodiment, the trafficking signal is derived from the Kir2.1 protein asspecified in Gradinaru et al. In another embodiment, an ER export motifis inserted at the C-terminus of the accessory fluorescent protein.

Targeting Mitochondria:

For measuring mitochondrial membrane potential or for studyingmitochondria, one may wish to localize PROPS to the mitochondrial innermembrane or mitochondrial outer membrane, in which case appropriatesignaling sequences can be added to the rhodopsin protein.

Optogenetic voltage sensors can be targeted to the inner mitochondrialmembrane, following a procedure such as that described in, e.g., A.Hoffmann, V. Hildebrandt, J. Heberle, and G. Büldt, “Photoactivemitochondria: in vivo transfer of a light-driven proton pump into theinner mitochondrial membrane of Schizosaccharomyces pombe,” Proc. Natl.Acad. Sci. USA 91, PNAS 9367-9371 (1994).

Codon Usage:

A large number of mammalian genes, including, for example, murine andhuman genes, have been successfully expressed in various host cells,including bacterial, yeast, insect, plant and mammalian host cells.Nevertheless, despite the burgeoning knowledge of expression systems andrecombinant DNA technology, significant obstacles remain when oneattempts to express a foreign or synthetic gene in a selected host cell.For example, translation of a synthetic gene, even when coupled with astrong promoter, often proceeds much more slowly than would be expected.The same is frequently true of exogenous genes that are foreign to thehost cell. This lower than expected translation efficiency is often dueto the protein coding regions of the gene having a codon usage patternthat does not resemble those of highly expressed genes in the host cell.It is known in this regard that codon utilization is highly biased andvaries considerably in different organisms and that biases in codonusage can alter peptide elongation rates. It is also known that codonusage patterns are related to the relative abundance of tRNAisoacceptors, and that genes encoding proteins of high versus lowabundance show differences in their codon preferences.

Codon-optimization techniques have been developed for improving thetranslational kinetics of translationally inefficient protein codingregions. These techniques are based on the replacement of codons thatare rarely or infrequently used in the host cell with those that arehost-preferred. Codon frequencies can be derived from literature sourcesfor the highly expressed genes of many organisms (see, for example,Nakamura et al., 1996, Nucleic Acids Res. 24: 214-215). Thesefrequencies are generally expressed on an ‘organism-wide average basis’as the percentage of occasions that a synonymous codon is used to encodea corresponding amino acid across a collection of protein-encoding genesof that organism, which are preferably highly expressed. In oneembodiment, the codons of a microbial rhodopsin protein are optimizedfor expression in a eukaryotic cell. In one embodiment, the eukaryoticcell is a human cell.

It is preferable but not necessary to replace all the codons of themicrobial polynucleotide with synonymous codons having highertranslational efficiencies in eukaryotic (e.g., human) cells than thefirst codons. Increased expression can be accomplished even with partialreplacement. Typically, the replacement step affects at least about 5%,10%, 15%, 20%, 25%, 30%, more preferably at least about 35%, 40%, 50%,60%, 70% or more of the first codons of the parent polynucleotide.Suitably, the number of, and difference in translational efficiencybetween, the first codons and the synonymous codons are selected suchthat the protein of interest is produced from the syntheticpolynucleotide in the eukaryotic cell at a level which is at least about110%, suitably at least about 150%, preferably at least about 200%, morepreferably at least about 250%, even more preferably at least about300%, even more preferably at least about 350%, even more preferably atleast about 400%, even more preferably at least about 450%, even morepreferably at least about 500%, and still even more preferably at leastabout 10000%, of the level at which the protein is produced from theparent polynucleotide in the eukaryotic cell.

Generally, if a parent polynucleotide has a choice of low andintermediate translationally efficient codons, it is preferable in thefirst instance to replace some, or more preferably all, of the lowtranslationally efficient codons with synonymous codons havingintermediate, or preferably high, translational efficiencies. Typically,replacement of low with intermediate or high translationally efficientcodons results in a substantial increase in production of thepolypeptide from the synthetic polynucleotide so constructed. However,it is also preferable to replace some, or preferably all, of theintermediate translationally efficient codons with high translationallyefficient codons for optimized production of the polypeptide.

Replacement of one codon for another can be achieved using standardmethods known in the art. For example codon modification of a parentpolynucleotide can be effected using several known mutagenesistechniques including, for example, oligonucleotide-directed mutagenesis,mutagenesis with degenerate oligonucleotides, and region-specificmutagenesis. Exemplary in vitro mutagenesis techniques are described forexample in U.S. Pat. Nos. 4,184,917, 4,321,365 and 4,351,901 or in therelevant sections of Ausubel et al. (Current Protocols in MolecularBiology, John Wiley & Sons, Inc. 1997) and of Sambrook et al.,(Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press,1989). Instead of in vitro mutagenesis, the synthetic polynucleotide canbe synthesized de novo using readily available machinery as described,for example, in U.S. Pat. No. 4,293,652. However, it should be notedthat the present invention is not dependent on, and not directed to, anyone particular technique for constructing the synthetic polynucleotide.

The genes for microbial rhodopsins (e.g., GPR) express well in E. coli,but less well in eukaryotic hosts. In one embodiment, to enableexpression in eukaryotes a version of the gene with codon usageappropriate to eukaryotic (e.g., human) cells is designed andsynthesized. This procedure can be implemented for any gene usingpublicly available software, such as e.g., the Gene Designer 2.0 package(available on the world wide web at dna20.com/genedesigner2/). Some ofthe “humanized” genes are referred to herein by placing the letter “h”in front of the name, e.g. hGPR. The Arch 3 rhodopsins and mutantsthereof described herein and in the examples are all optimized for humancodon usage.

Methods of Measuring Membrane Potential

Further provided herein are methods of measuring membrane potentialchanges in cells comprising the nucleic acid constructs or the vectorsprovided herein.

In certain embodiments, a method for measuring membrane potential in acell expressing a polynucleotide encoding the inventive polypeptidescomprises the steps of: a) exciting, in vitro, at least one cellcomprising a nucleic acid encoding an inventive polypeptide with lightof at least one wavelength; and b) detecting, in vitro, at least oneoptical signal from the at least one cell, wherein the level offluorescence emitted by the at least one cell compared to a reference isindicative of the membrane potential of the cell.

In certain embodiments, the inventive polypeptide is an archaerhodopsinvariant with reduced ion pumping activity compared to a naturalarchaerhodopsin from which it is derived and possesses improvedproperties as described herein.

In certain embodiments, the archaerhodopsin variant comprises a mutatedproton acceptor proximal to the Schiff Base. In certain embodiments, theat least one wavelength is a wavelength between 590 to 690 nm. Incertain embodiments, the at least one wavelength is a wavelength between600 to 690 nm. In certain embodiments, the at least one wavelength is awavelength between 620 to 690 nm. In certain embodiments, the at leastone wavelength is a wavelength between 640 to 690 nm. In certainembodiments, the at least one wavelength is a wavelength between 650 to690 nm.

In certain embodiments, the cell is a prokaryotic cell. In certainembodiments, the cell is a eukaryotic cell. In certain embodiments, theeukaryotic cell is a mammalian cell. In certain embodiments, theeukaryotic cell is a stem cell or a pluripotent or a progenitor cell. Incertain embodiments, the eukaryotic cell is an induced pluripotent cell.In certain embodiments, the eukaryotic cell is a neuron. In certainembodiments, the eukaryotic cell is a cardiomyocyte. In certainembodiments, the method comprises a plurality of cells.

In certain embodiments, the method comprises a step of transfecting, invitro, the at least one cell with a vector comprising thepolynucleotides encoding the inventive polypeptides herein. In certainembodiments, the method comprises the polynucleotides encoding theinventive polypeptides herein is operably linked to a cell-type specificpromoter. In certain embodiments, the method comprises thepolynucleotides encoding the inventive polypeptides herein is operablylinked to a membrane-targeting nucleic acid sequence. In certainembodiments, the membrane-targeting nucleic acid is a plasma membranetargeting nucleic acid sequence. In certain embodiments, themembrane-targeting nucleic acid sequence is a subcellularcompartment-targeting nucleic acid sequence. In certain embodiments, thesubcellular compartment is selected from a mitochondrial membrane, anendoplasmic reticulum, a sarcoplastic reticulum, a synaptic vesicle, anendosome and a phagosome.

In certain embodiments, the method comprises the polynucleotidesencoding the inventive polypeptides herein is operably linked to asecond polynucleotide sequence encoding at least one additionalfluorescent protein. In certain embodiments, the at least one additionalfluorescent protein is a fluorescent protein capable of indicating theion concentration in the cell. In certain embodiments, the fluorescentprotein capable of indicating ion concentration is a calcium indicator.In certain embodiments, the fluorescent protein capable of indicatingion concentration is a pH indicator.

In certain embodiments, the at least one additional fluorescent proteinis capable of undergoing nonradiative fluorescence resonance energytransfer to the inventive polypeptide, with a rate of energy transferdependent on the membrane potential. In certain embodiments, the atleast one additional fluorescent protein is GFP, YFP, citrine, mOrange2,mKate2, mRuby2, or a variant thereof.

In certain embodiments, the brightness of the fluorescent protein isinsensitive to membrane potential and local chemical environment.

In certain embodiments, the method further comprising steps of exciting,in vitro, the at least one cell with light of at least a first and asecond wavelength; and detecting, in vitro, the at least first and thesecond optical signal resulting from the excitation with the at leastthe first and the second wavelength from the at least one cell. Incertain embodiments, the at least second wave length is between 447-594nm. In certain embodiments, method further comprises a step ofcalculating the ratio of the fluorescence emission from the GEVIs to thefluorescence emission of the at least one additional fluorescent proteinto obtain a measurement of membrane potential independent of variationsin expression level.

In certain embodiments, the method further comprises the step ofexposing, in vitro, the at least one cell to a stimulus capable of, orsuspected to be capable of changing membrane potential.

In certain embodiments, the stimulus a candidate agent. In certainembodiments, the stimulus is a change to the composition of the cellculture medium.

In certain embodiments, the stimulus is an electrical current. Incertain embodiments, the method further comprises the step of measuring,in vitro, the at least one optical signal at a first and at least at asecond time point.

Cells

According to another aspect of the invention, a cell that expresses anyof the aforementioned embodiments of a vector or nucleic acid constructis provided. In another aspect, also provided are cells comprising theinventive polypeptides. Cells that are useful according to the inventioninclude eukaryotic and prokaryotic cells. Eukaryotic cells include cellsof non-mammalian invertebrates, such as yeast, plants, and nematodes, aswell as non-mammalian vertebrates, such as fish and birds. The cellsalso include mammalian cells, including human cells. The cells alsoinclude immortalized cell lines such as HEK, HeLa, CHO, 3T3, which maybe particularly useful in applications of the methods for drug screens.The cells also include stem cells, pluripotent cells, progenotir cells,and induced pluripotent cells. Differentiated cells including cellsdifferentiated from the stem cells, pluripotent cells and progenitorcells are included as well.

In some embodiments, the cells are cultured in vitro or ex vivo. In someembodiments, the cells are part of an organ or an organism.

The methods can also be applied to any other membrane-bound structure,which may not necessarily be classified as a cell. Such membrane boundstructures can be made to carry the microbial rhodopsin proteins of theinvention by, e.g., fusing the membranes with cell membrane fragmentsthat carry the microbial rhodopsin proteins of the invention.

Cells include also zebrafish cardiomyocytes; immune cells (primarymurine and human cultures and iPS-derived lines for all, in addition tothe specific lines noted below), including B cells (e.g., human Rajicell line, and the DT40 chicken cell line), T cells (e.g., human Jurkatcell line), Macrophages, Dendritic cells, and Neutrophils (e.g., HL-60line). Additionally, one can use glial cells: astrocytes andoligodendrocytes; pancreatic beta cells; hepatocytes; non-cardiac musclecells; endocrine cells such as parafollicular and chromaffin; and yeastcells. Cells also include neuronal cells, such as neurons, and skeletalcells.

The cell can also be a Gram positive or a Gram negative bacteria, aswell as pathogenic bacteria of either Gram type. The pathogenic cellsare useful for applications of the method to, e.g., screening of novelantibiotics that affect membrane potential to assist in destruction ofthe bacterial cell or that affect membrane potential to assistdestruction of the bacterial cell in combination with the membranepotential affecting agent; or in the search for compounds that suppressefflux of antibiotics.

The membrane potential of essentially any cell, or any phospholipidbilayer enclosed structure, can be measured using the methods andcompositions described herein. Examples of the cells that can be assayedare a primary cell e.g., a primary hepatocyte, a primary neuronal cell,a primary myoblast, a primary mesenchymal stem cell, primary progenitorcell, or it may be a cell of an established cell line. It is notnecessary that the cell be capable of undergoing cell division; aterminally differentiated cell can be used in the methods describedherein. In this context, the cell can be of any cell type including, butnot limited to, epithelial, endothelial, neuronal, adipose, cardiac,skeletal muscle, fibroblast, immune cells, hepatic, splenic, lung,circulating blood cells, reproductive cells, gastrointestinal, renal,bone marrow, and pancreatic cells. The cell can be a cell line, a stemcell, or a primary cell isolated from any tissue including, but notlimited to, brain, liver, lung, gut, stomach, fat, muscle, testes,uterus, ovary, skin, spleen, endocrine organ and bone, etc. Where thecell is maintained under in vitro conditions, conventional tissueculture conditions and methods can be used, and are known to those ofskill in the art. Isolation and culture methods for various cells arewell within the knowledge of one skilled in the art. The cell can be aprokaryotic or eukaryotic cell. In certain embodiments, the cell is amammalian cell. In certain embodiments, the cell is a human cell. In oneembodiment, the cell is a neuron or other cell of the brain. In someembodiment, the cell is a cardiomyocyte. In some embodiments, the cellis cardiomyocyte that has been differentiated from an inducedpluripotent cell.

Uses with Spectrally Orthogonal Polypeptides

The inventive polypeptides provided herein can be used alone or incombination with other polypeptides, such as blue-shifted opticalreporters or optical actuators. In certain embodiments, the polypeptidesprovided herein are used in combination with second polypeptide that isspectrally orthogonal, for example, another polypeptide that isexcitable with a different range of wavelengths such as blue light,thereby making the combination of the polypeptides useful as tools forall-optical electrophysiology. In certain embodiments, the thepolypeptides provided herein can be co-expressed with a bluelight-activated polypeptide. In certain embodiments, the twopolypeptides are co-expressed in the cell membrane. In certainembodiments, the second polypeptide is blue-shifted optical actuator.

For example, the inventive polypeptides used in combination with aspectrally orthogonal polypeptide would be useful to probe neuronalexcitation across spatial and temporal scales, for example, in cellularsystems ranging from single dendritic spines to fields containing dozensof neurons measured in parallel, and from microsecond delays associatedwith action potential propagation to days-long changes in excitability.

In certain embodiments, the polypeptides provided herein, alone or incombination with other polypeptides, are useful for studying theexcitability in human induced pluripotent stem cell (hiPSC)-derivedneurons and in tissue such as brain tissue.

Provided herein are methods for characterizing cellular physiology byincorporating into an electrically excitable cell an optical reporterof, and an optical actuator of, electrical activity. A signal isobtained from the optical reporter in response to a stimulation of thecell. Either or both of the optical reporter and actuator may be basedon genetically-encoded rhodopsins incorporated into the cell. Providedare all optical methods that may be used instead of, or as a complementto, traditional patch clamp technologies and that can provide rapid,accurate, and flexible assays of cellular physiology.

In certain embodiments, provided is a method for characterizing a cell,the method comprising incorporating into an electrically excitable cellan optical actuator of, and an optical reporter of, electrical activity;obtaining a signal from the optical reporter in response to astimulation of the cell; and evaluating the signal, therebycharacterizing the cell. In certain embodiments, provided is a methodfor characterizing a cell, the method comprising incorporating into anelectrically excitable cell an optical actuator of, and an opticalreporter of, electrical activity; obtaining a signal from the opticalreporter in response to a stimulation of the cell; and evaluating thesignal, thereby characterizing the cell, wherein the optical reporter isany one of the inventive polypeptides described herein.

In certain embodiments, the optical reporter is a polypeptide comprisingan amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequencecomprises at least one mutation selected from P60S, T80S, D95H, D106H,and F161V. In certain embodiments, the optical reporter is a polypeptidecomprising an amino acid sequence of SEQ ID NO: 1, wherein the aminoacid sequence comprises at least one mutation selected from P60S, T80S,D95Q, D106H, and F161V. In certain embodiments, the optical reporter isa polypeptide comprising an amino acid sequence of SEQ ID NO: 1, whereinthe amino acid sequence comprises a mutation at position 95, and atleast one mutation selected from P60S, T80S, D106H, and F161V. Incertain embodiments, the optical reporter is a polypeptide comprising anamino acid sequence of SEQ ID NO: 1, a D106H mutation, and at least onemutation selected from P60S, T80S, and F161V. In certain embodiments,the optical reporter is a polypeptide comprising an amino acid sequenceof SEQ ID NO: 2 or a sequence that is at least about 80% homologous oridentical to SEQ ID NO: 2. In certain embodiments, the optical reporteris a polypeptide comprising an amino acid sequence of SEQ ID NO: 3 or asequence that is at least about 80% homologous or identical to SEQ IDNO: 3. In certain embodiments, incorporating the actuator and reporterinto the cell comprises transforming the electrically active cell with avector that includes a nucleic acid encoding the optical actuator of,and the optical reporter of, electrical activity. In certainembodiments, the method further comprises obtaining a somatic cell andconverting the somatic cell into the electrically excitable cell.

In certain embodiments, converting the somatic cell into theelectrically active cell comprises one selected from the list consistingof: direct conversion; and via an iPS intermediary. In certainembodiments, the electrically excitable cell is derived from a humanembryonic stem cell. In certain embodiments, the electrically excitablecell is one selected from the list consisting of a neuron, acardiomyocyte, and a glial cell. In certain embodiments, the opticalactuator initiates an action potential in response to the stimulation.In certain embodiments, the stimulation comprises illuminating the cell.In certain embodiments, illuminating the cell is done using spatiallyresolved light from a digital micromirror array. In certain embodiments,the excitation of, and the signal from, the optical reporter compriselight that does not stimulate the cell. In certain embodiments,illuminating the cell and obtaining the signal are done simultaneously.

The optical actuator may be a genetically-encoded rhodopsin or modifiedrhodopsin such as a microbial channelrhodopsin. For example, sdChR, achannelrhodopsin from Scherffelia dubia, may be used or an improvedversion of sdChR—dubbed CheRiff—may be used as an optical actuator.“CheRiff” refers to a version of sdChR that uses mouse codonoptimization, a trafficking sequence, and the mutation E154A. CheRiff isa blue-light activated channelrhopdopsin (excitation peak of 474 nm).CheRiff has been described in U.S. patent application Ser. No.14/303,178, incorporated herein by reference in its entirety. Theoptical actuator generally carries current densities sufficient toinduce action potentials (APs) when only a subsection of a cell isexcited. For example, light used for imaging the reporter generally doesnot activate the actuator, and light used for activating the actuatorgenerally does not confound the fluorescence signal of the reporter.Thus in an embodiment, an optical actuator and an optical reporter arespectrally orthogonal to avoid crosstalk and allow for simultaneous use.

In certain embodiments, the optical actuator comprises a modifiedrhodopsin. In certain embodiments, the optical actuator comprisesCheRiff. In certain embodiments, the optical reporter comprises arhodopsin that has been modified for voltage-sensitive fluorescence andabsence of a steady-state photocurrent. In certain embodiments, theoptical reporter comprises an inventive polypeptide as described herein.

In certain embodiments, the method further comprises obtaining a controlcell and observing a control signal generated by a control opticalreporter in the control cell. In certain embodiments, obtaining thecontrol cell comprises editing a genome from the cell such that thecontrol cell and the cell are isogenic but for a mutation. In certainembodiments, obtaining the signal comprises observing a cluster ofdifferent cells with a microscope and using a computer to isolate thesignal generated by the optical reporter from a plurality of signalsfrom the different cells. In certain embodiments, the computer isolatesthe signal by performing an independent component analysis andidentifying a spike train associated with the cell. In certainembodiments, further comprising using the microscope to obtain an imageof a plurality of clusters of cells.

In certain embodiments, the observed signal comprises a probability of avoltage spike in response to the stimulation of the cell. In certainembodiments, the observed signal comprises a changed probability of avoltage spike in response to the stimulation of the cell relative to acontrol. In certain embodiments, the observed signal comprises a changein the waveform of a voltage spike. In certain embodiments, the observedsignal comprises a sub-threshold increase in the membrane potential. Incertain embodiments, the observed signal comprises a decrease in themembrane potential.

In certain embodiments, characterizing the cell comprises diagnosing adisease. In certain embodiments, the disease is selected from the groupconsisting of Cockayne syndrome, Down Syndrome, Dravet syndrome,familial dysautonomia, Fragile X Syndrome, Friedreich's ataxia, Gaucherdisease, hereditary spastic paraplegias, Machado-Joseph disease,Phelan-McDermid syndrome (PMDS), polyglutamine (polyQ)-encoding CAGrepeats, spinal muscular atrophy, Timothy syndrome, Alzheimer's disease,frontotemporal lobar degeneration, Huntington's disease, multiplesclerosis, Parkinson's disease, spinal and bulbar muscular atrophy, andamyotrophic lateral sclerosis.

In certain embodiments, characterizing the cell comprises evaluating aresponse of the cell to exposure to a compound. In certain embodiments,characterizing the cell further comprises measuring a concentration ofan ion. In certain embodiments, characterizing the cell comprisesdetermining progress of a treatment. In certain embodiments, the methodfurther comprising editing the genome of the electrically active cells.

Also provided herein is a method for characterizing an interactionbetween cells, the method comprising: incorporating into a firstelectrically excitable cell an optical actuator of electrical activityincorporating into a second electrically excitable cell an opticalreporter of electrical activity; culturing the first electricallyexcitable cell and the second electrically excitable cell in proximityto one another, obtaining a signal from the optical reporter in responseto a stimulation of the first electrically excitable cell; andevaluating the signal, thereby characterizing an interaction between thefirst electrically excitable cell and the second electrically excitablecell.

In certain embodiments, the first electrically excitable cell and thesecond electrically excitable cell are of the same cell type. In certainembodiments, the cell type is one selected from the list consisting of aneuron, a cardiomyocyte, and a glial cell.

In certain embodiments, the first electrically excitable cell and thesecond electrically excitable cell are each of a different cell type.

In certain embodiments, the characterized interaction comprisesexcitatory neurotransmission. In certain embodiments, the characterizedinteraction comprises inhibitory neurotransmission. In certainembodiments, characterizing the interaction comprises measurement ofconduction velocity of cardiac action potential. In certain embodiments,incorporating the actuator into the first electrically excitable cellcomprises transforming first electrically excitable cell with a vectorthat includes a nucleic acid encoding the optical actuator of electricalactivity.

In certain embodiments, incorporating the reporter into the secondelectrically excitable cell comprises transforming the secondelectrically excitable cell with a vector that includes a nucleic acidencoding the optical reporter of, electrical activity.

In certain embodiments, the method further comprising obtaining somaticcells and converting the somatic cells into the first electricallyexcitable cell and the second electrically excitable cell.

In certain embodiments, converting the somatic cells comprises oneselected from the list consisting of: direct conversion; and via an iPSintermediary. In certain embodiments, the first electrically excitablecell and the second electrically excitable cell are derived from a humanembryonic stem cell.

In certain embodiments, the optical actuator initiates an actionpotential in response to the stimulation. In certain embodiments, thestimulation comprises illuminating the first electrically excitablecell. In certain embodiments, the illuminating is done using spatiallyresolved light from a digital micromirror array. In certain embodiments,the excitation of, and the signal from, the optical reporter compriselight that does not stimulate the first electrically excitable cell. Incertain embodiments, the illuminating and obtaining the signal are donesimultaneously. In certain embodiments, the optical actuator comprises amodified rhodopsin. In certain embodiments, the optical actuatorcomprises CheRiff.

In certain embodiments, the optical reporter comprises a rhodopsin thathas been modified for voltage-sensitive fluorescence and absence of asteady-state photocurrent. In certain embodiments, the optical reportercomprises an inventive polypeptide as described herein.

In certain embodiments, obtaining the signal comprises observing acluster of different cells with a microscope and using a computer toisolate the signal generated by the optical reporter from a plurality ofsignals from the different cells.

In certain embodiments, the computer isolates the signal by performingan independent component analysis and identifying a spike trainassociated with the second electrically excitable cell. In certainembodiments, the method further comprising using the microscope toobtain an image of a plurality of clusters of cells.

In certain embodiments, the observed signal comprises a probability of avoltage spike in response to the stimulation of the cell. In certainembodiments, the observed signal comprises a changed probability of avoltage spike in response to the stimulation of the cell relative to acontrol. In certain embodiments, the observed signal comprises a changein the waveform of a voltage spike.

In certain embodiments, the observed signal comprises a sub-thresholdincrease in the membrane potential. In certain embodiments, the observedsignal comprises a decrease in the membrane potential.

In certain embodiments, characterizing the interaction comprisesdiagnosing a disease. In certain embodiments, characterizing theinteraction comprises evaluating a cellular response to exposure to acompound. In certain embodiments, characterizing the interactioncomprises determining progress of a treatment. In certain embodiments,the method further comprising editing the genome of the electricallyactive cells.

Other Uses of the Inventive Polypeptides

The polypeptides provided herein are useful for studying bioelectricphenomena such as neuronal or cardiac activity. For example, theproteins are useful in reporting action potentials in cultured neurons.

The constructs disclosed in the present application can be used inmethods for drug screening, e.g., for drugs targeting the nervous systemor for agents that affect the membrane potential of one or more of theintracellular membranes. In a culture of cells expressing specific ionchannels, one can screen for agonists or antagonists without the laborof applying patch clamp to cells one at a time. In neuronal cultures onecan probe the effects of drugs on action potential initiation,propagation, and synaptic transmission. Application in human inducedpluripotent stem cells (iPSC)-derived neurons will enable studies ongenetically determined neurological diseases, as well as studies on theresponse to environmental stresses (e.g., anoxia).

Similarly, the optical voltage sensing using the constructs providedherein provides a new and much improved methods to screen for drugs thatmodulate the cardiac action potential and its intercellular propagation.These screens will be useful both for determining safety of candidatedrugs and to identify new cardiac drug leads. Identifying drugs thatinteract with the hERG channel is a particularly promising directionbecause inhibition of hERG is associated with ventricular fibrillationin patients with long QT syndrome. Application in human iPSC-derivedcardiomyocytes will enable studies on genetically determined cardiacconditions, as well as studies on the response to environmental stresses(e.g., anoxia).

Additionally, the constructs of the present invention can be used inmethods to study of development and wound healing. The role ofelectrical signaling in normal and abnormal development, as well astissue repair, is poorly understood. VIPs enable studies of voltagedynamics over long times in developing or healing tissues, organs, andorganisms, and lead to drugs that modulate these dynamics.

In yet another embodiment, the invention provides methods to screen fordrugs that affect membrane potential of mitochondria. Mitochondria playan essential role in ageing, cancer, and neurodegenerative diseases.Currently there is no good probe for mitochondrial membrane potential.VIPs provide such a probe, enabling searches for drugs that modulatemitochondrial activity.

The invention further provides methods to screen for drugs that modulatethe electrophysiology of a wide range of medically, industrially, andenvironmentally significant microorganisms.

Prior to our discovery of VIPs, no measurement of membrane potential hadbeen made in any intact prokaryote. We discovered that bacteria havecomplex electrical dynamics. VIPs enable screens for drugs that modulatethe electrophysiology of a wide range of medically, industrially, andenvironmentally significant microorganisms. For instance, we found thatelectrical activity is correlated with efflux pumping in E. coli.

Changes in membrane potential are also associated with activation ofmacrophages. However, this process is poorly understood due to thedifficulty in applying patch clamp to motile cells. VIPs enable studiesof the electrophysiology of macrophages and other motile cells,including sperm cells for fertility studies. Thus the VIPs of theinvention can be used in methods to screen for drugs or agents thataffect, for example, immunity and immune diseases, as well as fertility.

The examples describe expression of VIPs in rat hippocampal neurons,mouse HL-1 cardiomyocytes, and human iPS-derived cardiomyocytes. In allcell types, single action potentials (APs) were readily observed. Wetested the effects of drugs on the AP waveform.

For example, in one embodiment, the invention provides a method whereinthe cell expressing a microbial rhodopsin is further exposed to astimulus capable of or suspected to be capable of changing membranepotential.

Stimuli that can be used include candidate agents, such as drugcandidates, small organic and inorganic molecules, larger organicmolecules and libraries of molecules and any combinations thereof. Onecan also use a combination of a known drug, such as an antibiotic with acandidate agent to screen for agents that may increase the effectivenessof the one or more of the existing drugs, such as antibiotics.

The methods of the invention are also useful for vitro toxicityscreening and drug development. For example, using the methods describedherein one can make a human cardiomyocyte from induced pluripotent cellsthat stably express a modified archaerhodopsin wherein the protonpumping activity is substantially reduced or abolished. Such cells areparticularly useful for in vitro toxicity screening in drug development.

General Experimental Methods

The invention provides method for measuring membrane potential in a cellexpressing a nucleic acid encoding a microbial rhodopsin protein, themethod comprising the steps of (a) exciting at least one cell comprisinga nucleic acid encoding a microbial rhodopsin protein with light of atleast one wavelength; and (b) detecting at least one optical signal fromthe at least one cell, wherein the level of fluorescence emitted by theat least one cell compared to a reference is indicative of the membranepotential of the cell.

The term “reference” as used herein refers to a baseline value of anykind that one skilled in the art can use in the methods. In someembodiments, the reference is a cell that has not been exposed to astimulus capable of or suspected to be capable of changing membranepotential. In one embodiment, the reference is the same cell transfectedwith the microbial rhodopsin but observed at a different time point.

In the methods of the invention, the cells are excited with a lightsource so that the emitted fluorescence can be detected. The wavelengthof the excitation light depends on the fluorescent molecule. Forexample, the archerhodopsin constructs in the examples are all excitableusing light with wavelengths varying between 594 nm and 690 nm or 594 nmto 645 nm. Alternatively, the range may be between 630 nm to 645 nm. Forexample a commonly used Helium Neon laser emits at 632.8 nm and can beused in excitation of the fluorescent emission of these molecules.

In some embodiments a second light is used. For example, if the cellexpresses a reference fluorescent molecule or a fluorescent moleculethat is used to detect another feature of the cell, such a pH or Calciumconcentration. In such case, the second wavelength differs from thefirst wavelength. Examples of useful wavelengths include wavelengths inthe range of 447-594 nm, for example, 473 nm, 488 nm, 514 nm, 532 nm,and 561 nm.

The hardware and software needed to take maximal advantage of VIPsdepends on the type of assay, and can be easily optimized and selectedby a skilled artisan based on the information provided herein. Existinginstrumentation can be easily used or adapted for the detection of VIPs.The factors that determine the type of instrumentation include,precision and accuracy, speed, depth penetration, multiplexing andthroughput. A general discussion is provided in US Publication No.20130224756, incorporated herein by reference in its entirety.

The spectroscopic states of microbial rhodopsins are typicallyclassified by their absorption spectrum. However, in some cases there isinsufficient protein in a single cell to detect spectral shifts viaabsorbance alone. Any of the exemplary several optical imagingtechniques known in the art (see, e.g., US Publication No. 20130224756,incorporated herein by reference in its entirety) can be used to probeother state-dependent spectroscopic properties.

Uses and Applications of the Voltage-Indicating Proteins

Provided herein are areas in which the voltage-indicating proteins, thepolynucleotides, the nucleic acid constructs, the vectors, and cells canbe applied both in commercial and scientific endeavors.

The present invention can be useful in screening drugs. A recent articlereported that “Among the 100 top-selling drugs, 15 are ion-channelmodulators with a total market value of more than $15 billion.”(Molokanova, E. & Savchenko, A. Drug Discov. Today 13, 14-22 (2008)).However, searches for new ion-channel modulators are limited by theabsence of good indicators of membrane potential (Przybylo, M., et al.J. Fluoresc., 1-19 (2010)). In some embodiments, the optical sensorsdescribed herein are used to measure or monitor membrane potentialchanges in response to a candidate ion channel modulator. Such screeningmethods can be performed in a high throughput manner by simultaneouslyscreening multiple candidate ion channel modulators in cells.

The present invention can be useful with stem cells. Many geneticallydetermined diseases of the nervous system and heart lack good animalmodels. In some embodiments, the VIPs described herein are expressed instem cells, either induced pluripotent or stem cells isolated from cordblood or amniotic fluid, or embryonic stem cells derived from humans orfetuses known to carry or be affected with a genetic defect. In someembodiments, the embryonal stem cells are of non-human origin.Alternatively the VIPs are expressed in progeny of the stem cells,either progenitor cells or differentiated cell types, such as cardiac orneuronal cells. Expression of voltage indicators in these cell typesprovides information on the electrophysiology of these cells and theresponse of membrane potential to candidate agents or to changes inambient conditions (e.g., anoxia). Additionally, expression of VIPs instem cells enables studies of the differentiation and development ofstem cells into electrically active cell types and tissues.

Stem cells may be isolated and manipulated according to methods known toone skilled in the art. Patents describing methods of making and using,e.g., primate embryonic stem cells are described in, e.g., U.S. Pat.Nos. 7,582,479; 6,887,706; 6,613,568; 6,280,718; 6,200,806; and5,843,780. Additionally, for example, human cord blood derivedunrestricted somatic stem cells are described in U.S. Pat. No. 7,560,280and progenitor cells from wharton's jelly of human umbilical cord inU.S. Pat. No. 7,547,546.

Induced pluripotent stem cells may be produced by methods described, forexample, in U.S. Patent Application Publication No. 20110200568,European Patent Application Publication No. 01970446, and U.S. PatentApplication Publication No. US2008/0233610. Additional methods formaking and using induced pluripotent stem cells are also described inapplication U.S. Ser. No. 10/032,191, titled “Methods for cloningmammals using reprogrammed donor chromatin or donor cells,” and Ser. No.10/910,156, “Methods for altering cell fate.” These patent applicationsrelate to technology to alter the state of a cell, such as a human skincell, by exposing the cell's DNA to the cytoplasm of anotherreprogramming cell with differing properties. Detailed description ofthe reprogramming factors used in making induced pluripotent stem cells,including expression of genes OCT4, SOX2, NANOG, cMYC, LIN28 can also befound, for example, in PCT/US2006/030632.

Methods for differentiating stem cells or pluripotent cells intodifferentiated cells are also well known to one skilled in the art.

The present invention is also useful in brain imaging. The human brainfunctions by sending electrical impulses along its ˜10¹¹ neurons. Thesepatterns of firing are the origin of every human thought and action. Yetthere is currently no good way to observe large-scale patterns ofelectrical activity in an intact brain (Baker, B. J. et al. J. Neurosci.Methods 161, 32-38 (2007); Baker, B. J. et al. Brain Cell Biology 36,53-67 (2008)).

The VIPs can lead to unprecedented insights in neuroscience. The devicecan allow mapping of brain activity in patients and/or cells of patientswith psychiatric and neurological diseases, and in victims of traumaticinjuries or animal models modeling such diseases and injuries.

Optical imaging of neuronal activity can also form the basis forimproved brain-machine interfaces for people with disabilities. Forimaging in the brain, the VIP is administered by direct injection intothe site to be analyzed (with or without accompanying electroporation)or the VIP is delivered using a viral vector. Alternatively the opticalsensor may be administered through the formation of a transgenicorganism, or through application of the Cre-Lox recombination system.

The present invention also has uses in microbiology. Bacteria are hostto dozens of ion channels of unknown function (Martinac, B., et al.Physiol. Rev. 88, 1449 (2008)). Most bacteria are too small for directelectrophysiological measurements, so their electrical properties arealmost entirely unknown.

Upon expressing PROPS (see, e.g., US 2013/0224756, incorporated byreference in its entirety) in E. coli, it was found that E. coli undergoa previously unknown electrical spiking behavior. The data describedherein in the Examples section is the first report of spontaneouselectrical spiking in any bacterium. This result establishes theusefulness of voltage sensors in microbes.

Furthermore, the electrical spiking in E. coli was found to be coupledto efflux of a cationic membrane permeable dye. It is thus plausiblethat electrical spiking is correlated to efflux of other cationiccompounds, including antibiotics. VIPs may prove useful in screens forinhibitors of antibiotic efflux.

VIPs will unlock the electrophysiology of the millions of species ofmicroorganisms which have proven too small to probe via conventionalelectrophysiology. This information will be useful for understanding thephysiology of bacteria with medical, industrial, and ecologicalapplications.

The present invention is also useful in the area of mitochondria andmetabolic diseases. Mitochondria are membrane-bound organelles which actas the ATP factories in eukaryotic cells. A membrane voltage powers themitochondrial ATP synthase. Dysfunction of mitochondria has beenimplicated in a variety of neurodegenerative diseases, diabetes, cancer,cardiovascular disease, and aging. Thus there is tremendous interest inmeasuring mitochondrial membrane potential in vivo, although currentlyavailable techniques are severely limited (Verburg, J. & Hollenbeck, P.J. J. Neurosci. 28, 8306 (2008); Ichas, F., et al. Cell 89, 1145-1154(1997); Johnson, L. V., et al. Proc. Natl. Acad. Sci. U.S.A. 77, 990(1980)).

The exemplary VIPs described herein (PROPS) can be tagged with peptidesequences that direct it to the mitochondrial inner membrane (Hoffmann,A., et al. Proc. Nat. Acad. Sci. U.S.A. 91, 9367 (1994)) or themitochondrial outer membrane, where it serves as an optical indicator ofmitochondrial membrane potential.

The present invention is also useful for imaging purposes in cells, suchas human cells and vertebrate models (e.g., rat, mouse, zebrafish). Forexample, The membrane potential of a mammalian cell can be detectedusing the archaerhodopsin variants of the inventive polypeptides.

The present invention is also useful in gene delivery methods. Thepolynucleotides encoding the archaerhodopsin polypeptides of theinvention are introduced to the cell or organ or organism of interestusing routine gene delivery methods. They are administered to a subjectfor the purpose of imaging membrane potential changes in cells of asubject. In one embodiment, the optical sensors are introduced to thecell via expression vectors.

The various gene delivery methods currently being applied to stem cellengineering include viral and non viral vectors, as well as biologicalor chemical methods of transfection. The methods can yield either stableor transient gene expression in the system used.

The present invention can also be used in viral gene delivery systems.Because of their high efficiency of transfection, genetically modifiedviruses have been widely applied for the delivery of genes into stemcells.

The present invention can also be used in DNA virus vectors, forexample, adenovirus and adeno-associated virus. Adenoviruses are doublestranded, nonenveloped and icosahedral viruses containing a 36 kb viralgenome (Kojaoghlanian et al., 2003). Their genes are divided into early(E1A, E1B, E2, E3, E4), delayed (IX, IVa2) and major late (L1, L2, L3,L4, L5) genes depending on whether their expression occurs before orafter DNA replication. More than 51 human adenovirus serotypes have beendescribed which can infect and replicate in a wide range of organs. Theviruses are classified into the following subgroups: A—induces tumorwith high frequency and short latency, B—are weakly oncogenic, and C—arenon-oncogenic (Cao et al., 2004; Kojaoghlanian et al., 2003).

These viruses have been used to generate a series of vectors for genetransfer cellular engineering. The initial generation of adenovirusvectors were produced by deleting the E1 gene (required for viralreplication) generating a vector with a 4 kb cloning capacity. Anadditional deletion of E3 (responsible for host immune response) allowedan 8 kb cloning capacity (Bett et al., 1994; Danthinne and Imperiale,2000; Danthinne and Werth, 2000). The second generation of vectors wasproduced by deleting the E2 region (required for viral replication)and/or the E4 region (participating in inhibition of host cellapoptosis) in conjunction with E1 or E3 deletions. The resultant vectorshave a cloning capacity of 10-13 kb (Armentano et al., 1995). The third“gutted” generation of vectors was produced by deletion of the entireviral sequence with the exception of the inverted terminal repeats(ITRs) and the cis acting packaging signals. These vectors have acloning capacity of 25 kb (Kochanek et al., 2001) and have retainedtheir high transfection efficiency both in quiescent and dividing cells.

Importantly, the adenovirus vectors do not normally integrate into thegenome of the host cell, but they have shown efficacy for transient genedelivery into adult stem cells. These vectors have a series ofadvantages and disadvantages. An important advantage is that they can beamplified at high titers and can infect a wide range of cells (Benihoudet al., 1999; Kanerva and Hemminki, 2005). The vectors are generallyeasy to handle due to their stability in various storing conditions.Adenovirus type 5 (Ad5) has been successfully used in delivering genesin human and mouse stem cells (Smith-Arica et al., 2003). The lack ofadenovirus integration into host cell genetic material can in manyinstances be seen as a disadvantage, as its use allows only transientexpression of the therapeutic gene.

The following provides examples to show that a skilled artisan canreadily transducer cells with contructs expressing microbial rhodopsinsof the present invention to eukaryotic, such as mammalian cells. Forexample in a study evaluating the capacity of mesenchymal stem cells toundergo chondrogenesis when TGF-beta1 and bone morphogencic protein-2(BMP-2) were delivered by adenoviral-mediated expression, thechondrogenesis was found to closely correlated with the level andduration of the transiently expressed proteins. Transgene expression inall aggregates was highly transient, showing a marked decrease after 7days. Chondrogenesis was inhibited in aggregates modified toexpress >100 ng/ml TGF-beta1 or BMP-2; however, this was partly due tothe inhibitory effect of exposure to high adenoviral loads (Mol. Ther.2005 August; 12 (2):219-28. Gene-induced chondrogenesis of primarymesenchymal stem cells in vitro. Palmer G D, Steinert A, Pascher A,Gouze E, Gouze J N, Betz O, Johnstone B, Evans C H, Ghivizzani S C). Ina second model using rat adipose derived stem cells transduced withadenovirus carrying the recombinant human bone morphogenic protein-7(BMP-7) gene showed promising results for an autologous source of stemcells for BMP gene therapy. However, activity assessed by measuringalkaline phosphatase in vitro was transient and peaked on day 8. Thusthe results were similar to those found in the chondrogenesis model(Cytotherapy. 2005; 7 (3):273-81).

Thus for experiments that do not require stable gene expressionadenovirus vectors is a good option.

Adenovirus vectors based on Ad type 5 have been shown to efficiently andtransiently introduce an exogenous gene via the primary receptor,coxsackievirus, and adenovirus receptor (CAR). However, some kinds ofstem cells, such as MSC and hematopoietic stem cells, cannot beefficiently transduced with conventional adenovirus vectors based on Adserotype 5 (Ad5), because of the lack of CAR expression. To overcomethis problem, fiber-modified adenovirus vectors and an adenovirus vectorbased on another serotype of adenovirus have been developed. (Mol.Pharm. 2006 March-April; 3 (2):95-103. Adenovirus vector-mediated genetransfer into stem cells. Kawabata K, Sakurai F, Koizumi N, Hayakawa T,Mizuguchi H. Laboratory of Gene Transfer and Regulation, NationalInstitute of Biomedical Innovation, Osaka 567-0085, Japan).

Such modifications can be readily applied to the use of the microbialrhodopsin constructs described herein, particularly in the applicationsrelating to stem cells.

Other applications include adeno-associated viruses (AAV), which areubiquitous, noncytopathic, replication-incompetent members of ssDNAanimal virus of parvoviridae family (G. Gao et al., New recombinantserotypes of AAV vectors. Curr Gene Ther. 2005 June; 5 (3):285-97). AAVis a small icosahedral virus with a 4.7 kb genome. These viruses have acharacteristic termini consisting of palindromic repeats that fold intoa hairpin. They replicate with the help of helper virus, which areusually one of the many serotypes of adenovirus. In the absence ofhelper virus they integrate into the human genome at a specific locus(AAVS1) on chromosome 19 and persist in latent form until helper virusinfection occurs (Atchison et al., 1965, 1966). AAV can transduce celltypes from different species including mouse, rat and monkey. Among theserotypes, AAV2 is the most studied and widely applied as a genedelivery vector. Its genome encodes two large opening reading frames(ORFs) rep and cap. The rep gene encodes four proteins Rep 78, Rep 68,Rep 52 and Rep 40 which play important roles in various stages of theviral life cycle (e.g. DNA replication, transcriptional control, sitespecific integration, accumulation of single stranded genome used forviral packaging). The cap gene encodes three viral capsid proteins VP1,VP2, VP3 (Becerra et al., 1988; Buning et al., 2003). The genomic 3′ endserves as the primer for the second strand synthesis and has terminalresolution sites (TRS) which serve as the integration sequence for thevirus as the sequence is identical to the sequence on chromosome 19(Young and Samulski, 2001; Young et al., 2000).

These viruses are similar to adenoviruses in that they are able toinfect a wide range of dividing and non-dividing cells. Unlikeadenovirus, they have the ability to integrate into the host genome at aspecific site in the human genome. Unfortunately, due to their ratherbulky genome, the AAV vectors have a limited capacity for the transferof foreign gene inserts (Wu and Ataai, 2000).

The present invention can be used in RNA virus vectors such asretroviruses and lentiviruses. Retroviral genomes consist of twoidentical copies of single stranded positive sense RNAs, 7-10 kb inlength coding for three genes; gag, pol and env, flanked by longterminal repeats (LTR) (Yu and Schaffer, 2005). The gag gene encodes thecore protein capsid containing matrix and nucleocapsid elements that arecleavage products of the gag precursor protein. The pol gene codes forthe viral protease, reverse transcriptase and integrase enzymes derivedfrom gag-pol precursor gene. The env gene encodes the envelopglycoprotein which mediates viral entry. An important feature of theretroviral genome is the presence of LTRs at each end of the genome.These sequences facilitate the initiation of viral DNA synthesis,moderate integration of the proviral DNA into the host genome, and actas promoters in regulation of viral gene transcription. Retroviruses aresubdivided into three general groups: the oncoretroviruses (MaloneyMurine Leukenmia Virus, MoMLV), the lentiviruses (HIV), and thespumaviruses (foamy virus) (Trowbridge et al., 2002).

Retroviral based vectors are the most commonly used integrating vectorsfor gene therapy. These vectors generally have a cloning capacity ofapproximately 8 kb and are generated by a complete deletion of the viralsequence with the exception of the LTRs and the cis acting packagingsignals.

The retroviral vectors integrate at random sites in the genome. Theproblems associated with this include potential insertional mutagenesis,and potential oncogenic activity driven from the LTR. The U3 region ofthe LTR harbors promoter and enhancer elements, hence this region whendeleted from the vector leads to a self-inactivating vector where LTRdriven transcription is prevented. An internal promoter can then be usedto drive expression of the transgene.

The initial studies of stem cell gene transfer in mice raised the hopethat gene transfer into humans would be equally as efficient (O'Connorand Crystal, 2006). Gene transfer using available retroviral vectorsystems to transfect multi-lineage long-term repopulating stem cells isstill significantly more efficient in the mouse.

Lentiviruses are members of Retroviridae family of viruses (M. Scherr etal., Gene transfer into hematopoietic stem cells using lentiviralvectors. Curr Gene Ther. 2002 February; 2 (1):45-55). They have a morecomplex genome and replication cycle as compared to the oncoretroviruses(Beyer et al., 2002). They differ from simpler retroviruses in that theypossess additional regulatory genes and elements, such as the tat gene,which mediates the transactivation of viral transcription (Sodroski etal., 1996) and rev, which mediates nuclear export of unspliced viral RNA(Cochrane et al., 1990; Emerman and Temin, 1986).

Lentivirus vectors are derived from the human immunodeficiency virus(HIV-1) by removing the genes necessary for viral replication renderingthe virus inert. Although they are devoid of replication genes, thevector can still efficiently integrate into the host genome allowingstable expression of the transgene. These vectors have the additionaladvantage of a low cytotoxicity and an ability to infect diverse celltypes. Lentiviral vectors have also been developed from Simian, Equineand Feline origin but the vectors derived from Human ImmunodeficiencyVirus (HIV) are the most common (Young et al., 2006).

Lentivirus vectors are generated by deletion of the entire viralsequence with the exception of the LTRs and cis acting packagingsignals. The resultant vectors have a cloning capacity of about 8 kb.One distinguishing feature of these vectors from retroviral vectors istheir ability to transduce dividing and non-dividing cells as well asterminally differentiated cells (Kosaka et al., 2004). The lentiviraldelivery system is capable of high infection rates in human mesenchymaland embryonic stem cells. In a study by Clements et al., the lentiviralbackbone was modified to express mono- and bi-cistronic transgenes andwas also used to deliver short hairpin ribonucleic acid for specificsilencing of gene expression in human stem cells. (Tissue Eng. 2006July; 12 (7):1741-51. Lentiviral manipulation of gene expression inhuman adult and embryonic stem cells. Clements M O, Godfrey A, CrossleyJ, Wilson S J, Takeuchi Y, Boshoff C).

The table below summarizes various characteristics of the viral vectors.

Insert Vector capacity genome Vector (kb) Tropism form ExpressionEfficiency Enveloped Retrovirus 8 Dividing Integrated Stable High cellsonly Lentivirus 8 Dividing Integrated Stable High and non- dividing Non-enveloped Adeno- <5 Dividing Episomal Stable High associated and non-and virus dividing integrated Adenovirus 2-24 Dividing EpisomalTransient High and non- dividing

The present invention can also be used in non-viral gene deliverysystems. For example, they are useful in methods for the facilitatedintegration of genes. In addition to the viral based vectors discussedabove, other vector systems that lack viral sequence can be used. Thealternative strategies include conventional plasmid transfer and theapplication of targeted gene integration through the use of integrase ortransposase technologies. These represent important new approaches forvector integration and have the advantage of being both efficient, andoften site specific in their integration. Currently three recombinasesystems are available for genetic engineering: cre recombinase fromphage P1 (Lakso et al., 1992; Orban et al., 1992), FLP (flippase) fromyeast 2 micron plasmid (Dymecki, 1996; Rodriguez et al., 2000), and anintegrase isolated from streptomyses phage I C31 (Ginsburg and Calos,2005). Each of these recombinases recognize specific target integrationsites. Cre and FLP recombinase catalyze integration at a 34 bppalindromic sequence called lox P (locus for crossover) and FRT (FLPrecombinase target) respectively. Phage integrase catalyzessite-specific, unidirectional recombination between two short attrecognition sites in mammalian genomes. Recombination results inintegration when the att sites are present on two different DNAmolecules and deletion or inversion when the att sites are on the samemolecule. It has been found to function in tissue culture cells (invitro) as well as in mice (in vivo).

The Sleeping Beauty (SB) transposon is comprised of two invertedterminal repeats of 340 base pairs each (Izsvak et al., 2000). Thissystem directs the precise transfer of specific constructs from a donorplasmid into a mammalian chromosome. The excision and integration of thetransposon from a plasmid vector into a chromosomal site is mediated bythe SB transposase, which can be delivered to cells as either in a cisor trans manner (Kaminski et al., 2002). A gene in a chromosomallyintegrated transposon can be expressed over the lifetime of a cell. SBtransposons integrate randomly at TA-dinucleotide base pairs althoughthe flanking sequences can influence integration.

Methods to Introduce or Deliver Vectors into Cells

There are various methods known in the art for introducing vectors intocells. For example, electroporation relies on the use of brief, highvoltage electric pulses which create transient pores in the membrane byovercoming its capacitance. One advantage of this method is that it canbe utilized for both stable and transient gene expression in most celltypes. The technology relies on the relatively weak nature of thehydrophobic and hydrophilic interactions in the phospholipid membraneand its ability to recover its original state after the disturbance.Once the membrane is permeabilized, polar molecules can be deliveredinto the cell with high efficiency. Large charged molecules like DNA andRNA move into the cell through a process driven by their electrophoreticgradient. The amplitude of the pulse governs the total area that wouldbe permeabilized on the cell surface and the duration of the pulsedetermines the extent of permeabilization (Gabriel and Teissie, 1997).The permeabilized state of the cell depends on the strength of thepulses. Strong pulses can lead to irreversible permeabilization,irreparable damage to the cell and ultimately cell death. For thisreason electroporation is probably the harshest of gene delivery methodsand it generally requires greater quantities of DNA and cells. Theeffectiveness of this method depends on many crucial factors like thesize of the cell, replication and temperature during the application ofpulse (Rols and Teissie, 1990).

The most advantageous feature of this technique is that DNA can betransferred directly into the nucleus increasing its likelihood of beingintegrated into the host genome. Even cells difficult to transfect canbe stably transfected using this method (Aluigi et al., 2005; Zerneckeet al., 2003). Modification of the transfection procedure used duringelectroporation has led to the development of an efficient gene transfermethod called nucleofection. The Nucleofector™ technology, is anon-viral electroporation-based gene transfer technique that has beenproven to be an efficient tool for transfecting hard-to-transfect celllines and primary cells including MSC (Michela Aluigi, Stem Cells Vol.24, No. 2, February 2006, pp. 454-461).

Biomolecule-based methods can also be used to introduce thepolypeptides, polynucleotides, nucleic acid constructs and vectors intocells. For example, protein transduction domains (PTD) are shortpeptides that are transported into the cell without the use of theendocytotic pathway or protein channels. The mechanism involved in theirentry is not well understood, but it can occur even at low temperature(Derossi et al. 1996). The two most commonly used naturally occurringPTDs are the trans-activating activator of transcription domain (TAT) ofhuman immunodeficiency virus and the homeodomain of Antennapediatranscription factor. In addition to these naturally occurring PTDs,there are a number of artificial peptides that have the ability tospontaneously cross the cell membrane (Joliot and Prochiantz, 2004).These peptides can be covalently linked to the pseudo-peptide backboneof PNA (peptide nucleic acids) to help deliver them into the cell.

Other delivery methods include the use of liposomes, which are syntheticvesicles that resemble the cell membrane. When lipid molecules areagitated with water they spontaneously form spherical double membranecompartments surrounding an aqueous center forming liposomes. They canfuse with cells and allow the transfer of “packaged” material into thecell. Liposomes have been successfully used to deliver genes, drugs,reporter proteins and other biomolecules into cells (Felnerova et al.,2004). The advantage of liposomes is that they are made of naturalbiomolecules (lipids) and are nonimmunogenic.

Diverse hydrophilic molecules can be incorporated into them duringformation. For example, when lipids with positively charged head groupare mixed with recombinant DNA they can form lipoplexes in which thenegatively charged DNA is complexed with the positive head groups oflipid molecules. These complexes can then enter the cell through theendocytotic pathway and deliver the DNA into lysosomal compartments. TheDNA molecules can escape this compartment with the help ofdioleoylethanolamine (DOPE) and are transported into the nucleus wherethey can be transcribed (Tranchant et al., 2004).

Despite their simplicity, liposomes suffer from low efficiency oftransfection because they are rapidly cleared by the reticuloendothelialsystem due to adsorption of plasma proteins. Many methods of stabilizingliposomes have been used including modification of the liposomal surfacewith oligosaccharides, thereby sterically stabilizing the liposomes (Xuet al., 2002).

Immunoliposomes are liposomes with specific antibodies inserted intotheir membranes. The antibodies bind selectively to specific surfacemolecules on the target cell to facilitate uptake. The surface moleculestargeted by the antibodies are those that are preferably internalized bythe cells so that upon binding, the whole complex is taken up. Thisapproach increases the efficiency of transfection by enhancing theintracellular release of liposomal components. These antibodies can beinserted in the liposomal surface through various lipid anchors orattached at the terminus of polyethylene glycol grafted onto theliposomal surface. In addition to providing specificity to genedelivery, the antibodies can also provide a protective covering to theliposomes that helps to limit their degradation after uptake byendogenous RNAses or proteinases (Bendas, 2001). To further preventdegradation of liposomes and their contents in the lysosomalcompartment, pH sensitive immunoliposomes can be employed (Torchilin,2006). These liposomes enhance the release of liposomal content into thecytosol by fusing with the endosomal membrane within the organelle asthey become destabilized and prone to fusion at acidic pH.

In general non-viral gene delivery systems have not been as widelyapplied as a means of gene delivery into stem cells as viral genedelivery systems. However, promising results were demonstrated in astudy looking at the transfection viability, proliferation anddifferentiation of adult neural stem/progenitor cells into the threeneural lineages neurons. Non-viral, non-liposomal gene delivery systems(ExGen500 and FuGene6) had a transfection efficiency of between 16%(ExGen500) and 11% (FuGene6) of cells. FuGene6-treated cells did notdiffer from untransfected cells in their viability or rate ofproliferation, whereas these characteristics were significantly reducedfollowing ExGen500 transfection. Importantly, neither agent affected thepattern of differentiation following transfection. Both agents could beused to genetically label cells, and track their differentiation intothe three neural lineages, after grafting onto ex vivo organotypichippocampal slice cultures (J Gene Med. 2006 January; 8 (1):72-81.Efficient non-viral transfection of adult neural stem/progenitor cells,without affecting viability, proliferation or differentiation. Tinsley RB, Faijerson J, Eriksson P S).

Polymer-based methods can also be used for delivery. The protonated.epsilon.-amino groups of poly L-lysine (PLL) interact with thenegatively charged DNA molecules to form complexes that can be used forgene delivery. These complexes can be rather unstable and showed atendency to aggregate (Kwoh et al., 1999). The conjugation ofpolyethylene glycol (PEG) was found to lead to an increased stability ofthe complexes (Lee et al., 2005, Harada-Shiba et al., 2002). To confer adegree of tissue-specificity, targeting molecules such astissue-specific antibodies have also been employed (Trubetskoy et al.,1992, Suh et al., 2001).

An additional gene carrier that has been used for transfecting cells ispolyethylenimine (PEI) which also forms complexes with DNA. Due to thepresence of amines with different pKa values, it has the ability toescape the endosomal compartment (Boussif et al., 1995). PEG graftedonto PEI complexes was found to reduce the cytotoxicity and aggregationof these complexes. This can also be used in combination with conjugatedantibodies to confer tissue-specificity (Mishra et al., 2004, Shi etal., 2003, Chiu et al., 2004, Merdan et al., 2003).

Targeted gene delivery (site-specific recombinations) are also useful indelivery. In certain embodiments, a non-human, transgenic animalcomprising a targeting vector that further comprises recombination sites(e.g., Lox sites, FRT sites) can be crossed with a non-human, transgenicanimal comprising a recombinase (e.g., Cre recombinase, FLP recombinase)under control of a particular promoter. It has been shown that thesesite-specific recombination systems, although of microbial origin forthe majority, function in higher eukaryotes, such as plants, insects andmice. Among the site-specific recombination systems commonly used, theremay be mentioned the Cre/Lox and FLP/FRT systems. The strategy normallyused consists of inserting the loxP (or FRT) sites into the chromosomesof ES cells by homologous recombination, or by conventionaltransgenesis, and then of delivering Cre (or FLP) for the latter tocatalyze the recombination reaction. The recombination between the twoloxP (or FRT) sites may be obtained in ES cells or in fertilized eggs bytransient expression of Cre or using a Cre transgenic mouse. Such astrategy of somatic mutagenesis allows a spatial control of therecombination because the expression of the recombinase is controlled bya promoter specific for a given tissue or for a given cell.

A detailed description of the FRT system can be found, e.g., in U.S.Pat. No. 7,736,897.

The P1 bacteriophage uses Cre-lox recombination to circularize andfacilitate replication of its genomic DNA when reproducing. Since beingdiscovered, the bacteriophage's recombination strategy has beendeveloped as a technology for genome manipulation. Because the cre geneand loxP sites are not native to the mouse genome, they are introducedby transgenic technology into the mouse genomes (Nagy A. 2000. Crerecombinase: the universal reagent for genome tailoring. Genesis26:99-109). The orientation and location of the loxP sites determinewhether Cre recombination induces a deletion, inversion, or chromosomaltranslocation (Nagy A. 2000. Cre recombinase: the universal reagent forgenome tailoring. Genesis 26:99-109). The cre/lox system has beensuccessfully applied in mammalian cell cultures, yeasts, plants, mice,and other organisms (Araki K, Imaizumi T, Okuyama K, Oike Y, Yamamura K.1997. Efficiency of recombination by Cre transient expression inembryonic stem cells: comparison of various promoters. J Biochem (Tokyo)122:977-82). Much of the success of Cre-lox is due to its simplicity. Itrequires only two components: (a) Cre recombinase: an enzyme thatcatalyzes recombination between two loxP sites; and (b) LoxP sites: aspecific 34-base pair bp) sequences consisting of an 8-bp core sequence,where recombination takes place, and two flanking 13-bp invertedrepeats.

Another method for delivery is cell-mediated delivery. In oneembodiment, the optical sensors of the present invention are deliveredusing e.g., a cell expressing the optical sensor. A variety of means foradministering cells to subjects are known to those of skill in the art.Such methods can include systemic injection, for example i.v. injectionor implantation of cells into a target site in a subject. Cells may beinserted into a delivery device which facilitates introduction byinjection or implantation into the subjects. Such delivery devices mayinclude tubes, e.g., catheters, for injecting cells and fluids into thebody of a recipient subject. In certain embodiments, the tubesadditionally have a needle, e.g., a syringe, through which the cells ofthe invention can be introduced into the subject at a desired location.The cells may be prepared for delivery in a variety of different forms.For example, the cells may be suspended in a solution or gel or embeddedin a support matrix when contained in such a delivery device. Cells maybe mixed with a pharmaceutically acceptable carrier or diluent in whichthe cells of the invention remain viable. Pharmaceutically acceptablecarriers and diluents include saline, aqueous buffer solutions, solventsand/or dispersion media. The use of such carriers and diluents is wellknown in the art. The solution is generally sterile and fluid.Generally, the solution is stable under the conditions of manufactureand storage and preserved against the contaminating action ofmicroorganisms such as bacteria and fungi through the use of, forexample, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, andthe like. Solutions of the invention may be prepared by incorporatingcells as described herein in a pharmaceutically acceptable carrier ordiluent and, as required, other ingredients enumerated above, followedby filtered sterilization. The mode of cell administration can berelatively non-invasive, for example by intravenous injection, pulmonarydelivery through inhalation, oral delivery, buccal, rectal, vaginal,topical, or intranasal administration.

However, the route of cell administration will depend on the tissue tobe treated and may include implantation or direct injection. Methods forcell delivery are known to those of skill in the art and can beextrapolated by one skilled in the art of medicine for use with themethods and compositions described herein. Direct injection techniquesfor cell administration can also be used to stimulate transmigrationthrough the entire vasculature, or to the vasculature of a particularorgan, such as for example liver, or kidney or any other organ. Thisincludes non-specific targeting of the vasculature. One can target anyorgan by selecting a specific injection site, such as e.g., a liverportal vein. Alternatively, the injection can be performed systemicallyinto any vein in the body. This method is useful for enhancing stem cellnumbers in aging patients. In addition, the cells can function topopulate vacant stem cell niches or create new stem cells to replenishthe organ, thus improving organ function. For example, cells may take uppericyte locations within the vasculature. Delivery of cells may also beused to target sites of active angiogenesis. If so desired, a mammal orsubject can be pre-treated with an agent, for example an agent isadministered to enhance cell targeting to a tissue (e.g., a homingfactor) and can be placed at that site to encourage cells to target thedesired tissue. For example, direct injection of homing factors into atissue can be performed prior to systemic delivery of ligand-targetedcells.

Method of using stem cells, such as neural stem cells to deliver agentsthrough systemic administration and via intracranial administration tohome in on a tumor or to an injured parts of brain have been described(see, e.g., U.S. Pat. Nos. 7,655,224; and 7,393,526). Accordingly, onecan also modify such cells to express the desired voltage sensor fordelivery into the organs, such as the brain.

Membrane fusion reactions are common in eukaryotic cells. Membranes arefused intracellularly in processes including endocytosis, organelleformation, inter-organelle traffic, and constitutive and regulatedexocytosis. Intercellularly, membrane fusion occurs during sperm-eggfusion and myoblast fusion. Further discussion of membrane usionmediated delivery of an optical sensor is provided in US Publication No.2013/0224756, incorporated by reference.

Examples of other expression vectors and host cells are the pET vectors(Novagen), pGEX vectors (Amersham Pharmacia), and pMAL vectors (NewEngland labs. Inc.) for protein expression in E. coli host cells such asBL21, BL21(DE3) and AD494(DE3)pLysS, Rosetta (DE3), and Origami(DE3)(Novagen); the strong CMV promoter-based pcDNA3.1 (Invitrogen) andpCIneo vectors (Promega) for expression in mammalian cell lines such asCHO, COS, HEK-293, Jurkat, and MCF-7; replication incompetent adenoviralvector vectors pAdeno X, pAd5F35, pLP-Adeno-X-CMV (Clontech),pAd/CMVN5-DEST, pAd-DEST vector (Invitrogen) for adenovirus-mediatedgene transfer and expression in mammalian cells; pLNCX2, pLXSN, andpLAPSN retrovirus vectors for use with the Retro-X™ system from Clontechfor retroviral-mediated gene transfer and expression in mammalian cells;pLenti4N/V5-DEST™, pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ(Invitrogen) for lentivirus-mediated gene transfer and expression inmammalian cells; adenovirus-associated virus expression vectors such aspAAV-MCS, pAAV-IRES-hrGFP, and pAAV-RC vector (Stratagene) foradeno-associated virus-mediated gene transfer and expression inmammalian cells; BACpak6 baculovirus (Clontech) and pFastBac™ HT(Invitrogen) for the expression in Spodopera frugiperda 9 (Sf9) and Sfl1insect cell lines; pMT/BiPN/V5-His (Invitrogen) for the expression inDrosophila Schneider S2 cells; Pichia expression vectors pPICZα, pPICZ,pFLDα and pFLD (Invitrogen) for expression in Pichia pastoris andvectors pMETα and pMET for expression in P. methanolica; pYES2/GS andpYDI (Invitrogen) vectors for expression in yeast Saccharomycescerevisiae. Recent advances in the large scale expression heterologousproteins in Chlamydomonas reinhardtii are described by Griesbeck C. et.al. 2006 Mol. Biotechnol. 34:213-33 and Fuhrmann M. 2004, Methods MolMed. 94:191-5. Foreign heterologous coding sequences are inserted intothe genome of the nucleus, chloroplast and mitochondria by homologousrecombination. The chloroplast expression vector p64 carrying theversatile chloroplast selectable marker aminoglycoside adenyltransferase (aadA), which confers resistance to spectinomycin orstreptomycin, can be used to express foreign protein in the chloroplast.The biolistic gene gun method can be used to introduce the vector in thealgae. Upon its entry into chloroplasts, the foreign DNA is releasedfrom the gene gun particles and integrates into the chloroplast genomethrough homologous recombination.

Cell-free expression systems are also contemplated. Cell-free expressionsystems offer several advantages over traditional cell-based expressionmethods, including the easy modification of reaction conditions to favorprotein folding, decreased sensitivity to product toxicity andsuitability for high-throughput strategies such as rapid expressionscreening or large amount protein production because of reduced reactionvolumes and process time. The cell-free expression system can useplasmid or linear DNA. Moreover, improvements in translation efficiencyhave resulted in yields that exceed a milligram of protein permilliliter of reaction mix. An example of a cell-free translation systemcapable of producing proteins in high yield is described by Spirin A S.et. al., Science 242:1162 (1988). The method uses a continuous flowdesign of the feeding buffer which contains amino acids, adenosinetriphosphate (ATP), and guanosine triphosphate (GTP) throughout thereaction mixture and a continuous removal of the translated polypeptideproduct. The system uses E. coli lysate to provide the cell-freecontinuous feeding buffer. This continuous flow system is compatiblewith both prokaryotic and eukaryotic expression vectors. As an example,large scale cell-free production of the integral membrane protein EmrEmultidrug transporter is described by Chang G. el. al., Science310:1950-3 (2005). Other commercially available cell-free expressionsystems include the Expressway™ Cell-Free Expression Systems(Invitrogen) which utilize an E. coli-based in-vitro system forefficient, coupled transcription and translation reactions to produce upto milligram quantities of active recombinant protein in a tube reactionformat; the Rapid Translation System (RTS) (Roche Applied Science) whichalso uses an E. coli-based in-vitro system; and the TNT CoupledReticulocyte Lysate Systems (Promega) which uses a rabbitreticulocyte-based in-vitro system.

It is understood that the foregoing detailed description and thefollowing examples are illustrative only and are not to be taken aslimitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments, which will be apparent tothose skilled in the art, may be made without departing from the spiritand scope of the present invention. Further, all patents, patentapplications, and publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments are based on the information available to the applicants anddo not constitute any admission as to the correctness of the dates orcontents of these documents.

EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

Experimental Methods

Engineering of Arch. We adopted a hierarchical approach to screeningthat prioritized brightness over multiple secondary selection criteria.The brightness screen was conducted by examining the fluorescence oflarge libraries of variants expressed in bacterial colonies. Subsequentscreens for trafficking, speed, and voltage sensitivity were performedin HeLa cells subjected to field stimulation and induced transmembranevoltages, and then in HEK cells with patch clamp.

Molecular biology procedures: Synthetic DNA oligonucleotides used forcloning and library construction were purchased from Integrated DNATechnologies. Pfu polymerase (Fermentas) or AccuPrime Pfx SuperMix(Invitrogen) were used for high fidelity non-mutagenic PCRamplifications in the buffer supplied by the respective manufacturer.Taq polymerase (New England Biolabs) in the presence of MnCl₂ (0.1 mM)was used for error-prone PCR. PCR products and products of restrictiondigests were routinely purified using preparative agarose gelelectrophoresis followed by DNA isolation using the GeneJET gelextraction kit (Fermentas). Restriction endonucleases were purchasedfrom Fermentas and used according to the manufacturer's recommendedprotocol. Ligations were performed using T4 ligase (Invitrogen) orGibson Assembly (New England Biolabs). Small-scale isolation of plasmidDNA was performed by GeneJET miniprep kit (Fermentas). The cDNAsequences for all Arch variants and fusion constructs were confirmed bydye terminator cycle sequencing using the BigDye Terminator CycleSequencing kit (Applied Biosystems). Site-directed mutagenesis andrandomization of targeted codons was performed with either theQuikChange Lightning Single or Multi kit (Agilent Technologies).

Construction of Arch mutant libraries: A library of >10⁴ mutants wasgenerated by error-prone PCR of the gene encoding Arch D95N. Thesevariants were then joined with the gene encoding mOrange2 by a two-partoverlap extension PCR. The 5′ piece used in the overlap extension wasprepared by error-prone PCR of Arch D95N as template with a mixture ofthe forward primer (Fw_XbaI_Arch) and reverse primer (RV_Arch). PrimerFw_XbaI_Arch contains an XbaI site and primer RV_Arch contains anoverlap region with primer FW_Arch_FP. The 3′ piece for use in theoverlap extension was prepared by high fidelity PCR amplification ofmOrange2 using a forward primer (FW_Arch_FP) and a reverse primer(RV_HindIII_FP). Primer RV_HindIII_FP contains a stop codon and aHindIII site. The full-length Arch-mOrange2 gene library was assembledby overlap extension PCR using an equimolar mixture of primersFw_XbaI_Arch and RV_HindIII_FP together with a mixture of the 5′ and 3′PCR fragments described above (50 ng each) as the template. In laterrounds of directed evolution, error-prone PCR and StEP PCR DNAshuffling⁶⁰ were both used for construction of Arch-mOrange2 genelibraries.

The full-length PCR product (approximately 1,500 b.p.) was purified byagarose gel electrophoresis, doubly digested, and ligated between theXbaI and HindIII sites of a modified pBAD vector which was generated bydeleting the ETorA tag between the NcoI and XbaI sites of the pTorPEvector⁶¹ using Quikchange Lightning kit.

Following ligation, electrocompetent E. coli strain DH10B wastransformed with the library of gene variants and cultured overnight at37° C. on 10-cm Petri dishes of LB-agar supplemented with 100 μL of 4 mMretinal (Sigma-Aldrich), 100 g/mL ampicillin (Sigma), and up to 0.0020%(wt/vol) L-arabinose (Alfa Aesar). The retinal solution was added on thesurface of LB-agar plates evenly and air-dried prior to plating the cellsuspension. At concentrations of L-arabinose higher than 0.0020%(wt/vol) we observed abnormal colony morphologies and reducedfluorescent brightness, presumably due to cytotoxicity caused byoverexpression.

Screening of Arch mutants in E. coli: The imaging system used forlibrary screening has previously been described in detail.⁶² We screened10,000-20,000 colonies (10-20 plates of bacterial colonies) per round ofrandom mutagenesis. For libraries generated by randomization of one ormore codons, we screened approximately 3-fold more colonies than theexpected library diversity (e.g. 3,000 colonies for a 1,000-memberlibrary).

We acquired two images of colonies using filter sets for mOrange2 (exc.540-580 nm, em. 600-660 nm) and Arch (exc. 604-640 nm and em. 660-700nm). An image of the ratio of Arch:mOrange2 fluorescence was calculated,and the colonies with the top 0.01% to 0.1% highest ratios were manuallypicked. Picked clones were individually cultured in 2 mL liquid LBmedium (200 μg/mL ampicillin) shaken (250 rpm) overnight at 37° C.

Protein expression was induced by adding 2 mL, of liquid LB mediumcontaining 120 μM retinal, 200 μg/mL ampicillin and 0.2% L-arabinose tothe overnight culture, followed by incubation at 37° C. for 3 hours. Thecell pellets were collected by centrifugation, washed and resuspended inbuffered M9 salt solution containing 7 g/L Na₂HPO₄, 3 g/L KH₂PO₄, 0.5g/L NaCl and 1 g/L NH₄Cl. The suspension was then diluted 5-fold priorto acquisition of its fluorescence spectrum in a Safire 2 fluorescencemicroplate reader (Tecan).

The emission profiles of each variant under excitation at 525 nm and 600nm were acquired and normalized by the absorbance at 600 nm. The cellpellets of the three variants with the highest ratios of Arch tomOrange2 and the two variants with the brightest absolute Archfluorescence were treated for plasmid DNA extraction, and the pooledgenes were used as templates for construction of gene libraries in thenext round of directed evolution.

After five iterations we arrived at a non-pumping variant of Arch withfive mutations relative to wild-type (P60S, T80S, D95N, D106Y, and F161V) and significantly improved brightness under excitation with lowillumination intensity. This variant, designated Arch 3.5, was used asthe template for subsequent efforts to address the secondary selectioncriteria.

Random mutagenesis at positions Asp95 and Asp106: We next focused ontuning other properties of Arch including voltage sensitivity, responsekinetics, membrane trafficking and the undesirable dependence ofbrightness on illumination intensity. Positions Asp95 and Asp106 of Archare structurally aligned with positions Asp85 and Asp96 ofbacteriorhodopsin, and have been reported to play key roles in protontranslocation during the photocycle.^(63, 64) The voltage sensingmechanism of Arch is likely due to electric-field-dependent protonationof the retinal Schiff base,^(10,65) so we reasoned that perturbations ofthe proton translocation network around the Schiff base couldpotentially affect the voltage sensitivity, response kinetics, orcomplex photophysics.²⁴

We constructed libraries in which Asp95 and Asp106 were randomized to asubset of all possible amino acid substitutions. First, we randomizedposition 95 using codon HVS (where H=A, C or T; V=A, C, or G; S═C or G),which encodes for all amino acids except Ala, Gly, Asp, Glu and Val.This library was screened by fluorescence imaging of E. coli colonies.Variants that retained a high ratio of Arch to mOrange2 fluorescencewere picked and expressed in HeLa cells for screening via inducedtransmembrane voltage (see below).

The mutation N951H emerged as the best from the first round of screeningin HeLa cells. We then constructed a second library by randomizingposition 106 to a subset of amino acids with polar or charged sidechains (codon NRC, where N=A, C, G, or T; R=A or G), and screened thesein HeLa cells. The variant with histidine at position 106 proved mostpromising and was designated QuasAr1.

Solubilization and spectroscopic characterization of QuasAr1 and QuasAr:E. coli expressing QuasAr1 and QuasAr2 were grown in 12 mL liquid LBmedium with 200 μg/ml ampicillin overnight. The next day, 12 mL ofliquid LB medium containing 50 μM retinal, 200 μg/ml ampicillin and 0.1%arabinose was added into the overnight culture, followed by additionalincubation at 37° C. for 4 hours. The cell pellets were collected bycentrifugation and lysed by suspension in B-PER solution (Pierce). Thecytoplasmic fraction was discarded after centrifugation and the coloredinsoluble fraction was resuspended in phosphate buffered saline (PBS)containing 1.5% n-dodecyl-3-D-maltopyranoside (Affymetrix, Inc.). Thesuspension was homogenized by an ultrasonic homogenizer and centrifuged(17,000 g for 15 mins, 4° C.). The solubilized protein in thesupernatant was used for in vitro spectroscopic characterization.

Absorption spectra were recorded on a DU-800 UV-visiblespectrophotometer (Beckman) and fluorescence spectra were recorded on aSafire2 plate reader (Tecan). Cy5 carboxylic acid (Cyandye) was used asthe reference for quantum yield measurement. Quantum yield measurementswere performed on a series of dilutions of each protein solution andstandard, with absorbance values ranging from 0.01 to 0.05 at 600 nm.The fluorescence emission spectra of each dilution were recorded withexcitation at 600 nm and the total fluorescence intensities obtained byintegration. Integrated fluorescence intensity vs. absorbance wasplotted for each protein and each standard. Quantum yields, Φ, weredetermined from the slopes (S) of each line using the equation:Φ_(protein)=Φ_(standard)×(S_(protein)/S_(standard)).

Expression vectors for HeLa cells: To express Arch-mOrange2 variants inHeLa cells, the gene in the pBAD vector was first amplified by PCR usingprimers Fw_BamHI_Kozak_Arch and RV_FP_ERex_stp_XbaI. This reverse primerencodes the endoplasmic reticulum (ER) export sequence from theinward-rectifier potassium channel Kir2.1 (FCYENE) (SEQ ID NO: 8),⁶⁶which has been reported to be effective for improving the membranetrafficking of Arch in mammalian cells.²⁵

The purified DNA was digested with BamHI and XbaI restriction enzymesand ligated into a purified pcDNA3.1 plasmid that had been digested withthe same two enzymes. The ligation reaction was used for transformationof electrocompetent E. coli strain DH10B cells. Cells were plated onLB/agar supplemented with ampicillin and individual colonies were pickedinto 4 mL of LB/ampicillin following overnight incubation at 37° C.Liquid cultures were shaken at 250 rpm and 37° C. for 12-15 h and then asmall scale isolation of plasmid DNA was performed. Each gene inpcDNA3.1 was fully sequenced using T7_FW, and BGH_RV primers. Plasmidswere then used for cell transfection as described below

Induced transmembrane voltage (ITV) in HeLa cells: We co-expressedprospective Arch variants in HeLa cells with the inward rectifierpotassium channel, Kir2.1. Expression of Kir2.1 lowered the restingpotential to approximately −60 mV, close to the resting potential ofneurons.^(67,68) We reasoned that this effect would center the ITV closeto the physiologically relevant range.

HeLa cells were grown to 40-60% confluence on home-made 35 mm glassbottom dishes or 24-well glass bottom plates. Cells were transfectedwith 1 μg of plasmid DNA comprising a 1:1 mixture of Arch variant andKir2.1, using either 2 μL Turbofect (Thermo Scientific) or 2 μLLipofectamine 2000 (Invitrogen) according to the manufacturer'sinstructions. After 3 h incubation, the medium was exchanged to DMEMwith 10% fetal bovine serum. Cells were incubated for an additional 24 hat 37° C. in a CO₂ incubator. Immediately prior to imaging, cells werewashed twice with Hanks balanced salt solution (HBSS) and then 1 mL of20 mM HEPES buffered HBSS was added.

Cell imaging was performed with an inverted Eclipse Ti-E (Nikon)equipped with a Photometrics QuantEM 512SC camera, a 150 W mercury-xenonlamp (Hamamatsu), and a 10 mW 638 nm semiconductor diode laser(56ICS/S2669, Melles Griot CleanBeam) aligned just above the angle fortotal internal reflection. The filters were: 590-650 nm (excitation),668-738 nm (emission), and 666 nm (dichroic). Movies were acquired at 10ms/frame. The NIS-Elements Advanced Research software (Nikon) was usedfor microscope and camera control and data acquisition. A schematic ofthe setup is shown in FIG. 2.

To probe the response kinetics and voltage sensitivity, we used a pairof parallel platinum electrodes to apply a reproducible electric fieldacross the cell culture and induce transient asymmetries in the membranevoltage.⁶⁹ Platinum electrodes with a gap of 0.5 cm were mounted in acustom plastic support. The electrode pair was placed in the imagingdish or well, and voltage pulses from a 6824A 40V/25A DC Power Supply(HP/Agilent) were applied using waveforms generated by a pulse generatorPG 58A (Gould Advance Ltd). The typical waveform had square-wave pulseslasting 20 ms, and pulse amplitudes from 25-35 V. Fluorescence wasimaged at 100 Hz frame rate in 4×4 binning mode for 10 seconds. Duringeach voltage pulse, opposite sides of the cell showed oppositefluorescence transients. Typical fluorescence traces are shown in FIG.2.

Raw fluorescence traces were corrected for background autofluorescenceand photobleaching. The average voltage sensitivity (ΔF/F_(min)) andsignal-to-noise ratio of each Arch variant were compared to the bestvariant of the previous generation, and only the variants with equal orimproved performance were chosen as templates for the next round ofscreening.

Expression vectors for HEK cells and neurons: To enable more accurateelectrophysiological characterization via patch clamp in HEK cells andprimary neuron cultures, we cloned QuasAr1 into the BamHI/EcoRI sites oflentivirus vector FCK-Arch-GFP (Addgene: 22217). This vector contains aCaMKIIα promoter and a Woodchuck Hepatitis Virus PosttranscriptionalRegulatory Element (WPRE) after the 3′ end of the open reading frame.The Arch cDNA was generated by PCR using forward primerFW_BamHI_Kozak_Arch_ValSer and overlapping reverse primers RV_FP_TS andRV_TS_ERex_stp_EcoRI. These reverse primers introduce a traffickingsignal (TS) motif and ER export signal peptide sequence at theC-terminus of the protein.

Simultaneous electrophysiology and fluorescence in HEK cells: HEK293Tcells (ATCC CRL-11268) were cultured and transfected following standardprotocols.¹⁰ Briefly, HEK-293 cells were grown at 37° C., 5% CO₂, inDMEM supplemented with 10% FBS and penicillin-streptomycin. Plasmidswere transfected using Transit 293T (Mirus) following the manufacturer'sinstructions, and assayed 48 hours later. The day before recording,cells were re-plated onto glass-bottom dishes (MatTek) at a density of˜10,000 cells/cm².

Cells were supplemented with retinal by diluting stock retinal solutions(40 mM, DMSO) in growth medium to a final concentration of 5 μM, andthen returning the cells to the incubator for 0.5-1 hour. All imagingand electrophysiology were performed in Tyrode buffer (containing, inmM: 125 NaCl, 2.5 KCl, 3 CaCl₂, 1 MgCl₂, 10 HEPES, 30 glucose pH 7.3,and adjusted to 305-310 mOsm with sucrose). A gap junction blocker,2-aminoethoxydiphenyl borate (50 μM, Sigma), was added to eliminateelectrical coupling between cells.

Filamented glass micropipettes (WPI) were pulled to a tip resistance of5-10 MΩ, and filled with internal solution containing 125 mM potassiumgluconate, 8 mM NaCl, 0.6 mM MgCl2, 0.1 mM CaCl2, 1 mM EGTA, 10 mMHEPES, 4 mM Mg-ATP, 0.4 mM Na-GTP (pH 7.3); adjusted to 295 mOsm withsucrose. Pipettes were positioned with a Sutter MP285 manipulator.Whole-cell, voltage and current clamp recordings were acquired using anAxopatch 700B amplifier (Molecular Devices), filtered at 2 kHz with theinternal Bessel filter and digitized with a National InstrumentsPCIE-6323 acquisition board at 5-10 kHz. Data was only acquired from HEKcells having reversal potentials between −10 and −40 mV, accessresistance <25 MΩ and membrane resistance >0.5 GΩ.

Simultaneous whole-cell patch clamp recordings and fluorescencerecordings were acquired on a home-built, inverted epifluorescencemicroscope, described previously¹⁰ and described below in “Apparatus”.For step response measurements, voltage clamp electronics werecompensated 90-95%. We examined variants of QuasAr1 with mutations atposition 95 (Asn, Cys, Gin, His and Tyr) and position 106 (Arg, Asp,Asn, Cys, Glu, His, Lys and Tyr). These experiments confirmed thathistidine at position 106 provided undetectable photocurrent, and thebest combination of improved voltage sensitivity, and fast kinetics.Mutants with Gin, Cys, or Asn at position 95 exhibited better voltagesensitivity compared to QuasAr1, while retaining fast kinetics. Wedesignated the H95Q mutant QuasAr2.

Analysis of mutations in QuasAr1 and QuasAr2: We developed a structuralmodel of QuasAr1 (FIG. 3) based on homologous protein Arch-2 (PDB:2EI4)⁷⁰. Mutations T80S and F161V are located in the periphery of theprotein, while P60S is close to the Schiff base of the retinalchromophore. Given their location, we suspect that the T80S and F161Vsubstitutions are unlikely to have a direct impact on the photophysicalproperties of the protein, and are more likely to have a role inimproving the folding efficiency. In contrast, the close proximity ofthe P60S substitution to the Schiff base suggests that this mutation hasa more direct influence on the photophysical properties.

We compared the Arch double mutants Arch (D95H, D106H) (termed “HH”) andArch(D95Q, D106H) (termed “QH”) to the corresponding QuasAr1 and QuasAr2mutants do determine whether the mutations in the proton-transport chainwere sufficient to induce the improved sensor performance. QuasAr1 andQuasAr2 were both significantly brighter than the corresponding doublemutants (FIG. 4). Furthermore, the voltage sensitivity of the HH, QH,QuasAr1 and wild-type protein were comparable, and three-fold less thanthe sensitivity of QuasAr2. Speeds were similar between the QuasArvariants and the double mutants. Thus one or more of the three mutationsoutside the proton transport chain (P60S, T80S, F161 V) plays animportant role in the brightness and sensitivity of the QuasAr mutants.The constructs described in herein are available on Addgene.

Oligonucleotides used in directed evolution of QuasAr mutants. Thesequences, from top to bottom, correspond to SEQ ID NOs: 12-27.

Name Sequence Fw_XbaI_Arch CGACTCTAGAATGGACCCCATCGCTCTGCAGGCTGGTTACGACCTGCTGGGTGACGGC RV_Arch TGCTACTACCGGTCGGGGCTCGGGGGCCTC FW_Arch_FPGAGGCCCCCGAGCCCCGACCGGTAGTAGCAATGGTGAGCAA GGGCGAGGAG RV_HindIII_FPGATGAAGCTTTTACTT GTACAGCTCGTCCATGCCG FW_Arch_95XCTATTATGCCAGGTACGCCHVSTGGCTGTTTACCACCCCAC FW_Arch_106XCCCCACTTCTGCTGCTGNRCCTGGCCCTTCTCGCTAA FW_Arch_95NATTATGCCAGGTACGCCAATTGGCTGTTTACCACC FW_Arch_95CCTA TTA TGC CAG GTA CGC CTGTTG GCT GTT TAC CAC CCC AC FW_Arch_95QCTA TTA TGC CAG GTA CGC CCAGTG GCT GTT TAC CAC CCC AC FW_Arch_106CCCCCACTTCTGCTGCTGTGCCTGGCCCTTCTCGCTAA Fw_Arch_106ECCCCACTTCTGCTGCTGGAGCTGGCCCTTCTCGCTAA Fw_BamHI_Kozak_ArchCGACGGATCCACCATGGACCCCATCGCTCTGCAGGC RV_FP_ERex_stp_XbaIGATGTCTAGATTATTCATTCTCATAACAAAACTTGTACAGCTCG TCCATGCCGFW_BamHI_Kozak_Arch_ValSer TGGGATCCACCATGGTAAGTATCGCTCTGCAGGCTGGTTACRV_FP_TS ATCCAGGGGGATGTACTCGCCTTCGCTTGTGATTCTACTCTTG TACAGCTCGTCCATGCCGRV_TS_ER export_stop_EcoRI GATGGAATTCTTATACTTCATTCTCATAACAAAATCCACCTACATTTATGTCTATTTGATCCAGGGGGATGTACTCGCC

Other Applications.

In some applications one might wish to use the visible spectrum forother imaging modalities, e.g. for a reporter of Ca²⁺ or a GFPexpression marker. In such cases, it is inconvenient to have mOrange2fused to Arch. Removal of the eGFP tag from Arch resulted in poormembrane localization in neurons. To maintain the beneficial traffickingproperties of the eGFP tag while eliminating the eGFP fluorescence, wemutated the eGFP chromophore from TYG to GGG using site-directedmutagenesis (Agilent). This mutation has been reported to preservefolding of eGFP.⁷² We also made versions of the fusion protein in whichthe mOrange2 was mutated to a non-fluorescent form by the mutation TYGto TAG. Nucleic acid constructs for the fusion protein were incorporatedinto lentiviral vectors under the CaMKIIα promoter, adapted from Addgeneplasmid 22217.

Neuronal Culture and Gene Delivery.

All procedures involving animals were in accordance with the NationalInstitutes of Health Guide for the care and use of laboratory animalsand were approved by the Institutional Animal Care and Use Committee atthe institution at which they were carried out.

Primary neurons: Rat glial monolayers were prepared similarly toprevious literature.⁷³ Briefly, 10⁶ dissociated hippocampal cells fromP0 rat pups (Sprague Dawley, Tocris)⁷⁴ were plated on a 10 cm culturedish in glial medium GM, comprised of 15% FBS (Life), 0.4% (w/v)D-glucose, 1% glutamax (Life), 1% penicillin/streptomycin (Life) in MEM(Life). When the dish reached confluence (1-2 weeks), cells were splitusing trypsin onto Mattek dishes (Mattek P35G-1.5-14-C) coated withmatrigel (BD biosciences) at a density of (3500 cells/cm²). After ˜3-6days, glial monolayers were at or near confluence and the medium wasreplaced by GM with 2 μM cytarabine (cytosine-β-arabinofuranoside).Dishes were maintained in GM with 2 μM cytarabine until use. Dishes werediscarded if microglia or neurons were identified on the monolayers.

Hippocampal neurons from P0 rat pups were dissected and cultured inneurobasal-based medium (NBActiv4, Brainbits llc.) at a density of30,000-40,000 cm⁻² on the pre-established glial monolayers.⁷⁴ At one dayin vitro (DIV), cytarabine was added to the neuronal culture medium at afinal concentration of 2 μM to inhibit further glial growth.⁷⁵

Neurons were transfected on DIV7 with the QuasArs plasmids via thecalcium phosphate transfection method.⁷⁶ Measurements on neurons weretaken between DIV 13-18.

For TTX-induced homeostatic plasticity, primary neurons were transfectedvia the calcium phosphate method on DIV7. TTX (1 μM) was added on DIV16. Excitability was measured on DIV 18 in Tyrodes medium with synapticblockers (10 μM NBQX, 25 μM AP-V, 20 μM Gabazine).

hiPSC-derived neurons: Human iPSC-derived iCell neurons from CellularDynamics Inc. were thawed and resuspended in complete iCell NeuronMaintenance Medium (CM) following manufacturer protocols. Neurons werethen plated at a density 125,000/cm² on pre-established rat glialmonolayers grown on glass-bottomed dishes. Medium was replaced 24 hourspost plating with CM supplemented with 10 ng/mL BDNF (Peprotech).Thereafter, 50% media exchanges with CM were done every 5 days.

For TTX-induced homeostatic plasticity, hiPSC-derived neurons weretransfected via the calcium phosphate method on DIV17. TTX (1 μM) wasadded on DIV 26. Excitability was measured on DIV 28 in Tyrodes mediumwith synaptic blockers (10 μM NBQX, 25 μM AP-V, 20 μM Gabazine).

For KCl-induced homeostatic plasticity, hiPSC-derived neurons weretransfected on DIV 10. KCl (15 mM) was added from DIV 18 to DIV 21 (60h). Excitability was measured on DIV 21 in Tyrodes medium with synapticblockers (10 μM NBQX, 25 μM AP-V, 20 μM Gabazine).

Organotypic brain slice culture: Organotypic hippocampal slices cultureswere prepared from postnatal day 6-8 Sprague-Dawley rats as describedpreviously.⁷⁷ The brain was taken out and immediately placed in chilleddissection media. Transverse hippocampal slices were cut with 400 μmthickness and 4 to 6 slices were placed in a sterile culture plateinsert (Millicell-CM, Millipore) in 6-well plates containing prewarmedmedia. Slices were biolistically transfected with a Helios Gene Gun(BioRad) at 2 days in vitro (DIV 2). Bullets were prepared using 12.5 μgof 1.6 μm gold particles and 80-100 μg of plasmid DNA. Slices weremaintained until imaging at DIV 12-16.

Immediately prior to inverted imaging, slices were affixed to a nylonmesh weight and mounted upside down in a delta T brainslice adapter forinverted microscope imaging (Bioptechs). Artificial cerebrospinal fluid(ACSF) was bubbled with carbogen (95% O₂, 5% CO₂) and flowed over theslice at 1 mL/min at 23° C.

Electrophysiology in Neurons.

Measurements were performed on primary cultures at 13-15 DIV.Experiments were conducted in Tyrode's solution containing 125 mM NaCl,2.5 mM KCl, 3 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, 30 mM glucose (pH 7.3)and adjusted to 305-310 mOsm with sucrose. Prior to imaging, neuronswere incubated with 5 μM all-trans retinal for 30 minutes and thenwashed with Tyrode's solution.

Synaptic blockers were added to the imaging medium for measurements ofsingle-cell electrophysiology (Arch photocurrents, and single-cellexcitability). The blockers comprised NBQX (10 μM, Tocris),D(−)-2-amino-5-phosphonovaleric acid (AP5; 25 μM, Tocris), and gabazine(SR-95531, 20 μM, Tocris). For measurements of channelrhodopsinphotocurrents in neurons, TTX (1 μM, Tocris) was included along with thesynaptic blockers to prevent recruitment of voltage gated sodiumchannels. Patch clamp data was used if and only if access resistance was<25 MΩ, and did not vary over the experiment. Recordings were terminatedif membrane resistance changed by >10%. Experiments were performed at23° C. under ambient atmosphere unless otherwise noted.

Comparison of QuasArs to Arclight A242: Arclight A242 was prepared in anidentical lentiviral plasmid driven by a CaMKIIα promoter and wastransfected (DIV 7) in parallel with the QuasAr plasmids in pairedcultures. We used a standard Arclight imaging intensity of 10 W/cm² at488 nm. QuasAr expressing neurons were imaged at two intensities (300and 800 W/cm² at 640 nm). All recordings were made on the setupdescribed below (“Apparatus”) at a 1 kHz frame rate and 60×magnification. Due to its slow kinetics at room temperature (FIG. 9),Arclight recordings were made at 34° C. to enhance SNR and to matchpreviously published conditions.²³ QuasAr2 reported APs with comparableSNR at 23° C. and 34° C. (41±3, n=8 cells, 640 nm 300 W/cm²). Forcomparisons in organotypic brain slice, Arclight was imaged at 50 W/cm²on an upright microscope to enable simultaneous patch clamp stimulationand recordings. Recordings were made at a 1 kHz frame rate as describedbelow (“Apparatus”) and were acquired at 34° C.

Immunostaining.

Cultures were fixed immediately following data acquisition in a solutionof 4% paraformaldehyde and 4% sucrose (w/v) in PBS, pH 7.4 at roomtemperature for 8 minutes. Fixed cultures were then washed three timesin Dulbecco's PBS supplemented with Ca²⁺ and Mg²⁺ (DPBS), pH 7.4, priorto permeabilization and blocking in a solution of 0.1% (w/v) gelatin and0.3% Triton-X-100 (v/v) in PBS, pH 7.4 (GTB) for 12-48 hours at 4 C.

For experiments using the sub-frame interpolation algorithm, primarycultures were fixed and stained using primary mouse monoclonalanti-ankyrin G (NeuroMab clone N106/36; 1:500), primary rabbitmonoclonal anti-GFP (Abcam ab32146, lot YK011702CS, 1:1000), secondarygoat anti-rabbit AlexaFluor 488 conjugated (Abcam ab150077, 1:500), andsecondary goat anti-mouse AlexaFluor 647 conjugated (Abcam ab150115,1:500) antibodies.

For experiments on human iPSC derived neurons, cultures were incubatedwith primary mouse anti-human nuclear antigen antibody (Millipore MAB1281 clone 235-1, 1:500) in GTB overnight at 4° C., then washed threetimes in DPBS, and incubated with rabbit anti-GFP AlexaFluor 488conjugated (polyclonal, Life A21311, 1:300) and secondary antibodydonkey anti-mouse AlexaFluor 647 (Life A31571, 1:300) in GTB overnightat 4 C. Cultures were washed three times in DPBS prior to mounting inDAPI Fluoromount-G (Southern Biotech).

Apparatus.

Experiments were conducted on a home-built inverted fluorescencemicroscope, similar to the one described in the methods.¹⁰ Briefly,illumination from a red laser 640 nm, 140 mW (Coherent Obis 637-140 LX),was expanded and focused onto the back-focal plane of a 60× oilimmersion objective, numerical aperture 1.45 (Olympus 1-U2B616). Imagingof brain slices was performed with a 20× water-immersion objective,numerical aperture 1.0 (Zeiss W Plan-Apo).

Illumination from a blue laser 488 nm 50 mW (Omicron PhoxX) was sentthrough an acousto-optic modulator (AOM; Gooch and Housego48058-2.5-0.55-5 W) for rapid control over the blue intensity. The beamwas then expanded and modulated by a digital micromirror device (DMD)with 608×684 pixels (Texas Instruments LightCrafter). The DMD wascontrolled via custom software (Matlab) through a TCP/IP protocol. TheDMD chip was re-imaged through the objective onto the sample, with theblue and red beams merging via a dichroic mirror. Each pixel of the DMDcorresponded to 0.65 m in the sample plane. A 532 nm laser was combinedwith the red and blue beams for imaging of mOrange2. We wrote softwareto map DMD coordinates to camera coordinates, enabling precise opticaltargeting of any point in the sample.

To achieve precise optical stimulation of user-defined regions of aneuron, it was necessary to determine the mapping from pixels on the DMDto pixels on the camera. A uniform fluorescent film (exc. 488 nm, em.515 nm) was loaded into the microscope. The DMD projected an array ofdots of known dimensions onto the sample. The camera acquired an imageof the fluorescence. Custom software located the centers of the dots inthe image, and created an affine transformation to map DMD coordinatesonto camera pixel coordinates.

A dual-band dichroic (Chroma zt532/635rpc) separated fluorescence ofmOrange2 and Arch from excitation light. A 531/40 nm bandpass filter(Semrock FF01-531/40-25) and 495 nm longpass dichroic (SemrockFF495-Di03) was used for eGFP imaging, a 710/100 nm bandpass filter(Chroma, HHQ710/100) was used for Arch imaging, and a quad-band emissionfilter (Chroma ZET405/488/532/642m) was used for mOrange2 imaging andpre-measurement calibrations. A variable-zoom camera lens (Sigma 18-200mm f/3.5-6.3 II DC) was used to image the sample onto an EMCCD camera(Andor iXon⁺ DU-860), with 128×128 pixels. The variable zoom enabledimaging at a range of magnifications while maintaining the high lightcollection efficiency of the oil or water immersion objectives.

In a typical experimental run, images of mOrange2 and QuasArfluorescence were first acquired at full resolution (128×128 pixels).Data was then acquired with 2×2 pixel binning to achieve a frame rate of1,000 frames/s. For experiments with infrequent stimulation (once every5 s), the red illumination was only on from 1 s before stimulation to 50ms after stimulation to minimize photobleaching. Cumulative red lightexposure was typically limited to <5 min. per neuron, althoughcontinuous red light exposure for 30 minutes was well tolerated.

Low magnification wide-field imaging was performed with a custommicroscope system based around a 2×, NA 0.5 objective (Olympus MVX-2).Illumination was provided by six lasers 640 nm, 500 mW (Dragon Lasers635M500), combined in three groups of two. Illumination was coupled intothe sample using a custom fused silica prism, without passing throughthe objective. Fluorescence was collected by the objective, passedthrough an emission filter, and imaged onto a scientific CMOS camera(Hamamatsu Orca Flash 4.0). Blue illumination for channelrhodopsinstimulation was provided by a 473 nm, 1 W laser (Dragon Lasers),modulated in intensity by an AOM and spatially by a DMD (Digital LightInnovations DLi4130-ALP HS). The DMD was re-imaged onto the sample viathe 2× objective.

During an experimental run, we first acquired an image of a neuron usingwide-field illumination at 640 nm and Arch fluorescence and/or 532 nmand mO2 fluorescence. A user then selected one or more regions ofinterest on the image of the neuron, and specified a timecourse for theillumination in each region. The software mapped the user-selectedpixels onto DMD coordinates and delivered the illumination instructionsto the DMD.

Data Analysis.

Statistics: All error ranges represent standard error of the mean,unless otherwise specified. For two-sample comparisons of a singlevariable, data was tested for normality using the Shapiro-Wilks test. Ifthe data was detectably non-Gaussian, we performed a nonparametricMann-Whitney U test. Otherwise we performed a two-tailed student'st-test. Probabilities of the null hypothesis p<0.05 were judged to bestatistically significant.

Extracting fluorescence from movies: Fluorescence values were extractedfrom raw movies in one of two ways. One approach used the maximumlikelihood pixel weighting algorithm.¹⁰ Briefly, the fluorescence ateach pixel was correlated with the whole-field average fluorescence.Pixels that showed stronger correlation to the mean were preferentiallyweighted. This algorithm automatically found the pixels carrying themost information, and de-emphasized background pixels. This approach wasused for all experiments in cultured neurons. In images containingmultiple neurons, the segmentation was performed semi-automaticallyusing the independent components-based approach.⁷⁸

Alternatively, a user defined a region comprising the cell body andadjacent processes, and calculated fluorescence from the unweighted meanof pixel values within this region. With the improved trafficking of theQuasAr mutants compared to Arch, the maximum likelihood pixel-weightingalgorithm was only marginally superior to manual definition of an regionof interest (ROI) (FIG. 10). In measurements in brain slice,fluorescence was calculated from manually defined ROIs with equal pixelweighting and no background subtraction or correction forphotobleaching.

Precision of optically recorded AP timing: To determine the temporalprecision of the QuasAr indicators, we used the sub-frame interpolationalgorithm^(37,38) to infer the timing with which the fluorescencereached 70% of maximum at each AP, and compared to simultaneouslyacquired high time-resolution patch clamp recordings. Root-mean-square(r.m.s.) temporal jitter was 44 μs for QuasAr1 (n=97 APs) and 61 μs forQuasAr2 (n=99 APs). This jitter reflects the combined errors in timingintrinsic to the optical measurement (shot-noise and distortion of thewaveform by the reporter) and errors introduced by temporaldiscretization of the camera frames and the sub-frame interpolation.Thus optical recordings with QuasArs can determine spike timing withprecision much greater than the camera exposure time.

Sub-Frame Interpolation of AP Timing.

The sub-frame interpolation algorithm consists of a series ofcomputational image-processing steps. Each step may be modified toaccount for experiment-specific attributes of the data.

A neuron was induced to fire through repeated optical stimulation of auser-selected subcellular compartment (typically soma or dendrite). Wetypically observed 5% photobleaching over a 40 s acquisition.Photobleaching was typically dominated by non-specific backgroundfluorescence, rather than by photobleaching of QuasAr, and oftenphotobleaching did not follow a simple single-exponential decay. Thephotobleaching baseline was constructed from the whole-field intensityby a sliding minimum filter, followed by a sliding mean filter. Eachframe of the movie was then corrected by dividing by this baseline.

QuasAr fluorescence intensity F(t) was determined either by theregression algorithm¹⁰ or by whole-field average intensity. Bothprocedures gave similar results, with slightly better signal-to-noiseratio returned by the regression algorithm (FIG. 2).

Determination of spike times was performed iteratively. A simplethreshold-and-maximum procedure was applied to F(t) to determineapproximate spike times, {T₀}. Waveforms in a brief window bracketingeach spike were averaged together to produce a preliminary spike kernelK₀(t). We then calculated the cross-correlation of K₀(t) with theoriginal intensity trace F(t). Whereas the timing of maxima in F(t) wassubject to errors from single-frame noise, the peaks in thecross-correlation, located at times {T}, were a robust measure of spiketiming. A movie showing the mean AP propagation was constructed byaveraging movies in brief windows bracketing spike times {T}. Typically100-400 APs were included in this average. The AP movie had highsignal-to-noise ratio, but did not clearly show signal propagation.

We applied spatial and temporal linear filters to further decrease thenoise in AP movie. The spatial filter consisted of convolution with aGaussian kernel, typically with a standard deviation of 1 pixel. Thetemporal filter was based upon Principal Components Analysis (PCA) ofthe set of single-pixel time traces. The time trace at each pixel wasexpressed in the basis of PCA eigenvectors. Typically the first 5eigenvectors were sufficient to account for >99% of the pixel-to-pixelvariability in AP waveforms, and thus the PCA eigendecomposition wastruncated after 5 terms. The remaining eigenvectors representeduncorrelated shot noise. Projections of the movie onto the PCAeigenvectors only showed spatial features above noise for the first 5eigenvectors. To verify that the spatial and PCA filtering did notdistort the underlying AP waveforms, we compared mean AP waveforms insubcellular compartments before and after the smoothing steps. Weobserved no systematic deviations in the AP waveforms in the axon, soma,or dendrites.

The user then set a threshold depolarization to track (represented as afraction of the maximum fluorescence transient), and a sign for dV/dt(indicating rising or falling edge). We chose 50% maximal depolarizationon the rising edge. The filtered data was fit with a quadratic splineinterpolation and the time of threshold crossing was calculated for eachpixel. The sub-frame timing precision of the algorithm was calibrated bypatch clamp measurements. Optically induced APs were recordedsimultaneously via QuasAr1 fluorescence in the soma and by conventionalpatch clamp, also in the soma. The r.m.s. error in timing was 54 μs inthis instance, and did not show systematic bias at the frame boundaries.

The fits were converted into movies showing AP propagation as follows.Each pixel was kept dark except for a brief flash timed to coincide withthe timing of the user-selected AP feature at that pixel. The flashfollowed a Gaussian timecourse, with amplitude equal to the local APamplitude, and duration equal to the cell-average time resolution, σ.Frame times in the sub-frame interpolation movies were selected to be˜2-fold shorter than σ.

Occasionally it was possible to enhance the spatial resolution of thehigh temporal resolution movies by mapping the timing data onto a higherspatial resolution static image of fluorescence of QuasAr1. The pixelmatrix of the sub-frame interpolated movie was expanded to match thedimensions of the high resolution image and the amplitude at each pixelwas then set equal to the mean brightness at that pixel. AP initiationat the axon initial segment is visible in the first two frames.

Example 1: Directed Evolution and Engineering of an Arch-Based VoltageIndicator

We previously showed that Archaerhodopsin 3 (Arch) functions as a fastand sensitive voltage indicator.¹⁰ Arch has the furthest red-shiftedspectrum of any GEVI, giving it the unique property of little spectraloverlap with channelrhodopsin actuators and GFP-based reporters. Thus itis natural to pair Arch-based indicators with optogenetic actuators forcrosstalk-free all-optical electrophysiology.

However, wild-type Arch had some undesirable attributes for a reporter:it was very dim, and the brightness was a nonlinear function ofillumination intensity.²⁴ Illumination for imaging generated ahyperpolarizing photocurrent, which partially suppressed neural firing.The mutant Arch(D95N) did not pump, but its step response was dominatedby a 41 ms time constant, too slow to resolve action potential (AP)waveforms.

We sought to repair these defects in engineered mutants of Arch. Toaccommodate the multiple selection criteria, we adopted a hierarchicalscreen (FIG. 1A). Five rounds of brightness screening in E. coli andrandom mutagenesis on a library of >10⁴ Arch mutants resulted in abrighter Arch variant, containing 5 point-mutations (Methods). Furthersite-directed mutagenesis at known key residues improved voltagesensitivity and speed (FIG. 2), while membrane trafficking was improvedby the addition of endoplasmic reticulum (ER) export motifs and atrafficking sequence (TS).²⁵ Two promising mutants were named QuasArs(Quality superior to Arch). QuasAr1 comprised mutations at P60S, T80S,D95H, D106H, F161V and QuasAr2 comprised mutations at P60S, T80S, D95Q,D106H, F161V. FIG. 3 shows absorption, fluorescence excitation, andemission spectra of the solubilized QuasAr proteins. The fluorescencequantum yields of solubilized QuasAr1 and 2 were 19- and 10-foldenhanced, respectively, relative to the non-pumping voltage indicatorArch(D95N) (Table 5). Table 5 shows the quantum yields of Arch variantsmeasured in solubilized protein. Fluorescence emission spectra wererecorded with excitation at 600 nm. Details of sample preparation andmeasurement procedures are given in Materials and Methods.

TABLE 5 Quantum yield relative Protein Name Quantum yield to Arch D95NArch N/A* N/A* Arch D95N 4 × 10⁻⁴ 1 QuasAr1 8 × 10⁻³ 19 QuasAr2 4 × 10⁻³10 Arch D95H/D106H 2 × 10⁻³ 4.2 Arch D95H/D106H/P60S 5 × 10⁻³ 12 ArchD95H/D106H/F161V 5 × 10⁻³ 13 *Due to the low light intensities used todetermine QYs, fluorescence from Arch was not detected above baseline.

We compared the fluorescence, voltage sensitivity, and speed of theQuasArs to wild-type Arch in HEK cells, using epifluorescence microscopyand whole-cell patch clamp electrophysiology. Under low intensityillumination (640 nm, 500 mW/cm²), QuasAr1 was 15-fold brighter thanwild-type Arch, and QuasAr2 was 3.3-fold brighter (FIG. 1B; Methods).Neither mutant showed the optical nonlinearity seen in the wild-typeprotein, implying that fluorescence was a 1-photon process with thevoltage-sensitive transition occurring from the ground state. At highintensity (>100 W/cm²) QuasAr1 was 2.5-fold brighter than wild-typeArch, while the brightness of QuasAr2 and of wild-type Arch werecomparable.

Fluorescence of Arch, QuasAr1, and QuasAr2 increased nearly linearlywith membrane voltage between −100 mV and +50 mV (FIG. 1C).Sensitivities were (ΔF/F per 100 mV): 32±3% for QuasAr1 (n=5 cells; allstatistics are mean±s.e.m. unless specified) and 90±2% for QuasAr2 (n=6cells). The sensitivity of QuasAr2 is a significant improvement overboth Arch (40% per 100 mV) and Arch(D95N) (60% per 100 mV).

Steps in membrane voltage (−70 mV to +30 mV) induced rapid fluorescenceresponses in both mutants, which we quantified on a fast photomultiplier(FIG. 1D). At room temperature (23° C.) QuasAr1 had a step response timeconstant of 0.053±0.002 ms (n=6 cells), close to the 0.05 ms timeresolution of the electronics and significantly faster than the 0.6 msstep response of wild-type Arch.²⁴ QuasAr2 had a bi-exponential stepresponse with time constants of 1.2±0.1 ms (68%) and 11.8±1.5 ms (32%)(n=6 cells). At 34° C., the apparent speed of QuasAr1 remained at the0.05 ms resolution of the electronics, and the time constants of QuasAr2decreased to 0.30±0.05 ms (62%) and 3.2±0.4 ms (38%) (n=7 cells). Bothmutants had similar response times on rising and falling edges (Table6). Table 6 shows spectroscopic and kinetic properties of Arch mutantsand ArcLight. Brightness, response speed, and sensitivity were measuredin HEK293 cells. Brightness and voltage sensitivity were comparable at34° C. and 23° C. Fluorescence response time to a voltage step (−70 mVto +30 mV and +30 mV to −70 mV) are shown. Fluorescence emission spectrawere recorded with excitation at 600 nm. Photocurrent (pA) in neurons(300 W/cm², 640 nm) for Arch (WT) is 220±30 and 0 in both QuasAr1 andQuasAr2. Details of sample preparation and measurement procedures aregiven in the General Experimental Methods section.

TABLE 6 Brightness (λ_(exc) = τ_(up) (ms, τ_(down) (ms, 640 nm) −70 mVto +30 mV to Sensitivity 0.7 800 +30 mV) −70 mV) (ΔF/F per Mutant W/cm²W/cm² τ₁ τ₂ % τ₁ τ₁ τ₂ % τ₁ 100 mV) 23° C. Arch(WT) 1 4.0 0.6 NA NA 0.251.9 67% 40% QuasAr1 15.2 10.3 0.05 3.2 94% 0.07 1.9 88% 33% QuasAr2 3.43.4 1.2 11.8 68% 1.0 15.9 80% 90% Arclight 17.4 123 39% 68 121 24%−32%   A242 34° C. QuasAr2 0.3 3.2 62% 0.3 4.0 73% Arclight 12 72 78%21.5 NA 100%  A242

In cultured rat hippocampal neurons, wild-type Arch generatedphotocurrents of 220±30 pA (n=6 cells) under red illumination often usedfor imaging (640 nm, 300 W/cm²) and 140±25 pA under blue light used foroptogenetic stimulation (488 nm, 500 mW/cm²) (FIG. 4). These currentshyperpolarized cells by 25±4 mV and 19±3 mV, respectively. NeitherQuasAr1 nor QuasAr2 generated detectable photocurrent in neurons underred light (tested up to 900 W/cm²) or blue light (FIG. 4).

Fluorescence of QuasAr1 and QuasAr2 reported APs in cultured neuronswith high electrical and temporal precision (FIG. 1E-H). We evoked APsvia current injection from a patch pipette and recorded the fluorescenceresponses of QuasAr1 and 2 under two standard illumination intensities(640 nm; 300 W/cm², 800 W/cm²). FIG. 10 shows a typical cell and themethod used for extracting fluorescence from movies. In recordings at a1 kHz frame rate signal-to-noise ratios (SNRs) for single APs were 21±2(300 W/cm², n=6 cells) to 32±4 (800 W/cm², n=6 cells) for QuasAr1 (FIG.1E) and 41±4 (300 W/cm², n=12 cells) to 70±8 (800 W/cm², n=12 cells) forQuasAr2 (FIG. 1G). These SNRs correspond to equivalent electrical noiselevels of 3.0 to 4.3 mV (800 to 300 W/cm²) for QuasAr1, or 1.5 to 2.2 mV(800 to 300 W/cm²) for QuasAr2 (Methods).

QuasAr1 did not introduce detectable broadening in the opticallyrecorded AP waveform, acquired at a 2 kHz frame rate (FIG. 1F). At roomtemperature, QuasAr2 broadened the optically recorded AP by 650±150 μsrelative to the simultaneously recorded electrical waveform at 70%maximum depolarization (n=5 cells; mean±s.d.) (FIG. 1H). At 34° C.,QuasAr2 broadened the optically recorded AP by 180±120 As (n=5 cells;mean±s.d). Both probes reported AP peak times with <100 μs jitterrelative to simultaneously acquired patch clamp recordings (Methods).

Photostability is a concern with any voltage indicator, so we quantifiedthe stability of QuasArs under continuous illumination at standardimaging intensity (640 nm, 300 W/cm²). Photobleaching time constantswere 440 s for QuasAr1 and 1020 s for QuasAr2. We further tested for redlight-induced phototoxicity using QuasAr2 as the readout. Undercontinuous illumination at 300 W/cm², QuasAr2 reported APs with 100%fidelity for the 30 min duration of the experiment, with no detectablechange in AP width or waveform.

The QuasArs represent the fastest and most sensitive GEVIs reportedto-date. The 50 μs response time of QuasAr1 is more than 10-fold fasterthan the fastest previously reported GEVIs^(24,26) and is comparable tofast voltage-sensitive dyes. QuasAr1 opens the possibility of accuratemapping of AP waveforms for even the fastest-spiking neurons.²⁷ Theillumination for imaging QuasArs, while intense, is ˜5-fold lower thanrequired for imaging Arch or Arch(D95N),¹ yet the QuasArs report voltagewith greatly improved sensitivity and time resolution compared to thefirst generation of Arch-based GEVIs. From a signal-to-noiseperspective, QuasAr2 is superior to QuasAr1: the greater voltagesensitivity of QuasAr2 outweighs the greater brightness of QuasAr1. Froma temporal resolution perspective, QuasAr1 is superior. We recommendQuasAr2 for spike counting and measurement of sub-threshold events, andQuasAr1 for measurement of microsecond-precision AP waveforms andtiming.

Example 2: Comparison of QuasArs to Arclight

We compared the QuasArs to Arclight A242, a recently introducedGFP-based GEVI.²³ Photophysical comparisons were performed in HEK cells,and action potential comparisons were performed in matched neuronalcultures (Methods). Arclight showed voltage sensitivity of −32±3% ΔF/Fper 100 mV (n=7 cells; FIG. 9), comparable in magnitude to QuasAr1 and2.8-fold smaller than QuasAr2. Arclight showed bi-exponential kineticsin response to rising or falling voltage steps (FIG. 9, Table 6). Meanhalf-response times were 42±8 ms and 76±5 ms on rising and falling edgesat 23° C. (n=6 cells) and 11±1 and 17±2 ms on rising and falling edgesat 34° C. (n=7 cells). Under continuous illumination at standard imagingintensity (488 nm, 10 W/cm²)¹¹ Arclight photobleached with a timeconstant of 70 s. In cultured neurons, Arclight reported actionpotentials with an amplitude of ΔF/F=−2.7±0.5% (n=5 cells) and asingle-trial signal-to-noise ratio (SNR) of 8.8±1.6 when recorded at a 1kHz frame rate (488 nm, 10 W/cm²) (Methods). Arclight distorted the APwaveforms to have a width of 14.5±3.0 ms at 70% maximal fluorescencedeviation, compared to the true width of 1.3±0.1 ms simultaneouslyrecorded with a patch pipette.

The Arclight reporter can be imaged with ˜30-fold lower illuminationintensity than is required for the QuasArs, facilitating measurements onreadily available microscope systems. The QuasArs reported actionpotentials with 7 to 16-fold larger fractional fluorescence changes, 3to 8-fold higher SNR, 30 to 1000-fold higher temporal resolution, and 6to 15-fold greater photostability. Furthermore, the far red excitationof the QuasArs allow, in principle, combination with channelrhodopsinactuators. This is not possible with Arclight or other GFP-based GEVIs.

Example 3: Electrochromic Fluorescence Resonance Energy Transfer(eFRET)-Based GEVIs

Importance of Spectral Tunability in GEVIs.

The spectral range of GEVIs is important when combining GEVIs with otherGEVIs, other optical reporters, or optogenetic actuators. Having GEVIsof multiple colors enables multiplex voltage imaging when distinctstructures cannot be spatially resolved. For instance, to studyseparately excitatory and inhibitory neurons in intact tissue wouldrequire two colors of GEVIs. Furthermore, GFP-based GEVIs cannot bepaired with other GFP-based reporters, e.g. gCaMP reporters of Ca2⁺,⁹iGluSnFR reporter of glutamate,⁸⁰ Perceval reporter of ATP,⁸¹ Clomelionreporter of Cl⁻,⁸² Pyronic reporter of pyruvate.⁸³ GFP-based reportersalso experience severe optical crosstalk with all optogenetic actuators.Even the reddest channelrhodopsin variants retain ˜20% activation withblue light used for GFP excitation.⁸⁴

Spectral range is also important for imaging in intact tissue. Theexcitation and emission spectra of flavins, a major contributor to brainautofluorescence, overlap strongly with those of GFP.^(85,86)Consequently, a rule of thumb is that the signal-to-background ratio forGFP-based reporters is 10-fold lower in tissue than in cell culture.Redder reporters have significantly less background in brain tissue.Furthermore light scattering in tissue scales as λ^(−x), where x˜2.33¹⁶.Thus illumination at 640 nm propagates nearly 1.9-fold further intotissue than does illumination at 488 nm. While two-photon excitation ofGFP-based reporters enables even greater depth penetration, this comesat the cost of requiring serial scanning, and a complex optical setup.

DETAILED DESCRIPTION

We invented a means to combine the high speed and sensitivity ofArch-based GEVIs with the brightness and spectral range of conventionalfluorescent proteins. We use voltage-induced changes in the absorptionspectrum of the retinal chromophore in Arch to alter the degree ofnonradiative quenching of a closely fused fluorescent protein.Traditionally, fluorescence resonance energy transfer (FRET) is used tomeasure the physical distance between a donor and acceptor. We useelectrochromic shifts in the acceptor to alter the degree of spectraloverlap between the emission of the donor and the absorption of theacceptor, thus we call the phenomenon electrochromic FRET (eFRET) (FIG.6).

The electrochromic quencher was QuasAr2, which was shown to exhibit fastand sensitive changes in fluorescence in response to changes in membranevoltage. The changes in fluorescence likely arose from changes in theabsorption spectrum, so we reasoned that QuasAr2 would be an effectivetool for voltage-dependent quenching of an appended fluorescent protein.

We invented five fast and sensitive GEVIs based upon fusions spanningthe visible spectrum. We created a palette of eFRET constructs by fusingGFP, YFP, citrine, mOrange2, mKate2 and mRuby2 to the C-terminus ofQuasAr2 via a short linker. We expressed these constructs in HEK293cells and tested the voltage sensitivity and step response using manualpatch-clamp electrophysiology (FIG. 7).

We further characterized the most sensitive eFRET voltage sensors viatransient transfection in cultured rat hippocampal neurons. Constructsexhibited good trafficking to the plasma membrane (FIG. 8). Injection ofcurrent pulses via a patch pipette (500-600 pA, 5-10 ms, 5 Hz) inducedtrains of action potentials, which induced downward fluorescencetransients of 12% (YFP), 12% (mOrange2), 7% (mRuby2).

The signal-to-noise ratio (SNR) of fluorescence detection, defined asthe ratio of the peak amplitude to standard deviation of fluorescence atthe baseline, was 6.6 (YFP), 11.6 (mOrange2), and 10.5 (mRuby2). In allcases images were acquired at 1 kHz with 3 W/cm² illumination intensity.Previous measurements with QuasAr2 reported an SNR of 30-70 at the samebandwidth but with 800 W/cm² illumination. At 3 W/cm² illumination, thedirect fluorescence of QuasAr2 is not detectable for exposure timescompatible with detecting single action potentials.

This work opens the possibility of multicolor voltage imaging with GEVIsspanning the visible spectrum.

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EQUIVALENTS AND SCOPE

As used in this specification and the claims, articles such as “a,”“an,” and “the” may mean one or more than one unless indicated to thecontrary or otherwise evident from the context. Claims or descriptionsthat include “or” between one or more members of a group are consideredsatisfied if one, more than one, or all of the group members are presentin, employed in, or otherwise relevant to a given product or processunless indicated to the contrary or otherwise evident from the context.The invention includes embodiments in which exactly one member of thegroup is present in, employed in, or otherwise relevant to a givenproduct or process. The invention includes embodiments in which morethan one, or all of the group members are present in, employed in, orotherwise relevant to a given product or process.

Furthermore, the invention encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, anddescriptive terms from one or more of the listed claims is introducedinto another claim. For example, any claim that is dependent on anotherclaim can be modified to include one or more limitations found in anyother claim that is dependent on the same base claim. Where elements arepresented as lists, e.g., in Markush group format, each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements and/or features, certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein. It is alsonoted that the terms “comprising” and “containing” are intended to beopen and permits the inclusion of additional elements or steps. Whereranges are given, endpoints are included. Furthermore, unless otherwiseindicated or otherwise evident from the context and understanding of oneof ordinary skill in the art, values that are expressed as ranges canassume any specific value or sub-range within the stated ranges indifferent embodiments of the invention, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Because such embodimentsare deemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the invention can be excluded from any claim,for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

1-60. (canceled)
 61. A polynucleotide encoding a polypeptide having atleast 80% identity to SEQ ID NO: 1, wherein the polypeptide comprises,relative to SEQ ID NO: 1, at least a D95Q substitution mutation and atleast one substitution selected from the group consisting of P60S, T80S,D106H, and F161V.
 62. A nucleic acid construct comprising thepolynucleotide of claim
 61. 63. An expression vector comprising thepolynucleotide of claim
 61. 64. A cell comprising the polynucleotide ofclaim
 61. 65. (canceled)
 66. A cell comprising the vector of claim 63.67. A kit comprising the polypeptide or variant thereof, polynucleotideor variant, nucleic acid construct, vector, or cell of any one of thepreceding claims.
 68. A method for measuring membrane potential in acell expressing a nucleic acid encoding a microbial rhodopsin protein,the method comprising the steps of: exciting, in vitro, at least onecell comprising the polynucleotide of claim 61 with light of at leastone wave length; and detecting, in vitro, at least one optical signalfrom the at least one cell, wherein the level of fluorescence emitted bythe at least one cell compared to a reference is indicative of themembrane potential of the cell.
 69. An expression vector comprising apolynucleotide encoding: a polypeptide having at least 80% identity toSEQ ID NO: 1, wherein the polypeptide comprises, relative to SEQ ID NO:1, at least one mutation selected from the group consisting of P60S,T80S, D95H, D95Q, D106H, and F161V; and a promoter sequence to controlthe expression of the polypeptide.
 70. The expression vector of claim69, wherein the at least one mutation is selected from the groupconsisting D95H and D95Q and the polypeptide further comprises, relativeto SEQ ID NO: 1, at least a second mutation selected from the groupconsisting of P60S, T80S, D106H, and F161V.
 71. The expression vector ofclaim 70, wherein the at least one mutation is D95Q.
 72. The expressionvector of claim 71, wherein the polypeptide comprises an amino sequenceof SEQ ID NO.
 3. 73. The expression vector of claim 70, wherein thepolypeptide comprises an amino sequence of SEQ ID NO.
 2. 74. Theexpression vector of claim 70, wherein the polynucleotide furtherencodes a second polypeptide.
 75. The expression vector of claim 74,wherein the second polypeptide is fluorescent.
 76. The expression vectorof claim 75, wherein the second polypeptide is mOrange2.
 77. Theexpression vector of claim 74, wherein the second polypeptide is capableof indicating ion concentration.